Multiple antigen presenting system (maps)-based staphylococcus aureus vaccine, immunogenic composition, and uses thereof

ABSTRACT

The present embodiments provide for an  S. aureus  (SA) Multiple Antigen Presenting System (MAPS) immunogenic composition comprising an immunogenic polysaccharide which induces an immune response, where at least one  S. aureus  (SA) peptide or polypeptide antigen is associated to the immunogenic polysaccharide by complementary affinity molecules. In some embodiments, the immunogenic polysaccharide can be an antigenic capsular polysaccharide of a Type 5 or Type 8 from  S. aureus , or alternatively, a different immunogenic capsular or noncapsular polysaccharide, and where the protein or peptide SA antigens are indirectly linked via an affinity binding pair. The present SA-MAPS immunogenic compositions can elicit both humoral and cellular immune responses to the immunogenic polysaccharide and one or multiple SA antigens at the same time.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.16/498,856 filed on Sep. 27, 2019, which is a 35 U.S.C. § 371 NationalPhase Entry Application of International Application No.PCT/US2018/024810 filed Mar. 28, 2018, which designates the U.S. andclaims benefit of priority to U.S. Provisional Application 62/477,618filed Mar. 28, 2017, the contents of each are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 18, 2022, isnamed 701039-088361-USCi_SL.txt and is 83,311 bytes in size.

FIELD OF THE INVENTION

The present invention relates to molecular genetics, immunology, andmicrobiology. The present application is generally directed tocompositions and methods for preparation of immunogenic compositions.More specifically, an embodiment of the present invention provides foran immunogenic composition comprising at least one immunogenicStaphylococcus aureus protein or peptide antigen attached to animmunogenic polysaccharide. In some embodiments, this complex can beused as an immunogenic composition, such as a vaccine, to confer asynergistic humoral and cellular immune response; and in someembodiments, elicits synergistic antibody and/or B-cell response andalso in some embodiments, a T-cell mediated protection against S. aureusinfection and colonization and carriage.

BACKGROUND OF INVENTION

Staphylococcus aureus (SA) is an important Gram-positive bacterium thatcauses a wide range of infections in both healthy and compromisedindividuals. SA is one of the leading causes of community- andhospital-acquired bacterial infections and postsurgical woundinfections, resulting in prolonged hospital stay and significantlyincreased healthcare cost. Staphylococcal bacteremia is associated withhigh mortality (about 20-40% in adults) even after appropriateantibiotic treatment. Skin and soft tissue infection (SSTI) is a commonchronic SA infection with frequent recurrence. Depending on the severityand depth of the infection, SSTI may represent as scalded skin syndrome,boils, impetigo, cellulitis, abscess, fasciitis or myonecrosis. SA isalso a cause of invasive disease, including meningitis, endocarditis,osteomyelitis, pneumonia, sepsis and toxic shock syndrome. SA colonizesabout 20% of the human population persistently and up to 80%transiently, serving as a reservoir for future infection andtransmission. The treatment of SA infection includes surgical procedure,antibiotics, or a combination of both. However, the effectiveness ofantibiotic treatment has been severely impacted by the rapid emergenceof multi-drug resistant strains (Methicillin-resistant SA, MRSA, as wellas Vancomycin-intermediate strains, or VISA) in both community-acquired(CA−) and hospital-acquired (HA−) infections in the past two decades.

Humans are the natural reservoirs for Staphylococcus aureus (S. aureus).Healthy individuals can be colonized by S. aureus on the skin, in thenares and the throat either persistently (10-35%), intermittently(20-75%) or be in a non-carriage state (5-70%) with no associateddisease. See Vandenbergh et al, J. Clin. Micro. 37:3133-3140 (1999).Disease subsequently occurs when individuals become immunocompromiseddue to breaches in immune barriers, such as during surgery, placement ofindwelling catheters or other devices, trauma, or wounds. The resultingS. aureus infection can cause a wide range of diseases that range frommild skin infections to endocarditis, osteomyelitis, bacteremia, sepsis,and other forms of disease with accompanying high mortality rates. Thelarge human reservoir enhances opportunity for evolution and spread ofadapted pathogenic clonal types.

Invasive staphylococcal infections from the Gram positive cocci S.aureus and S. epidermidis are of particular concern because they are anincreasing public health problem worldwide. Specifically, S. aureus isresponsible for the majority of hospital-acquired (nosocomial)infections, and its prevalence in community-onset infections isincreasing. For example, the incidence of invasive methicillin-resistantS. aureus (MRSA) was estimated at 31.8 per 100,000 persons, including18,650 deaths in the United States in 2005. See Klevens R. M. et al,JAMA, 298: 1763-71 (2007). Staphylococcal diseases have seen a dramaticincrease in the last 20 years; this increase parallels the use ofintravascular devices and invasive procedures. The rise in diseaseincidence is made more troubling because of the parallel rise ofantibiotic resistance; therefore, there is an urgent need forimmunogenic compositions for use in vaccines or to elicit polyclonal ormonoclonal antibodies to confer passive immunity as a means to preventor treat staphylococcal infection and associated diseases.

A vaccine against SA would represent a very attractive alternative.Vaccines provide prevention of and treatment for a variety of diseases,including microorganism infection, viral infection, and cancers. Successof polysaccharide-based vaccines and passive immunization for theprevention of colonization or disease has demonstrated the importance ofcapsular antibodies, in particular in controlling disease caused by S.pneumoniae. Further, studies in both animals and humans demonstrate thatantibodies elicited from pneumococcal vaccination can protect againstnasopharyngeal (NP) pneumococcal colonization, which precedespneumococcal disease.

If successful, a SA vaccine could provide broad, long-term benefit tothe population via both direct and herd immunities. Efforts in the earlySA vaccine development have focused on generating antibodies to variouspolysaccharide or protein antigens, including the capsularpolysaccharides, the extracellular polysaccharides, the toxins and thesurface proteins. The strategy of taking a combination of capsularpolysaccharides and/or proteins has been successfully used against manyhuman pathogens, such as Haemophilus influenzae type b, Streptococcuspneumoniae, Neisseria meningitidis (including most recently serogroupB), pertussis. The same approach has been attempted for vaccines for SA.However, unfortunately, to date, all the vaccine candidates for SA,which include use of SA polysaccharides and proteins in vaccines, orantibodies directed against these antigens, have failed in clinicaltrials. This is not expected considering that there was cleardemonstration of efficacy of these vaccines in various animal models ofinvasive SA infections.

Given this failure, there remains a need to improve the efficacy of SAvaccines, particularly to prevent infection and/or colonization andcarriage.

SUMMARY OF THE INVENTION

The present invention provides for an immunogenic multiple antigenpresenting system (MAPS) comprising an immunogenic polysaccharide, andattached to the immunogenic polysaccharide via an affinity binding pair,at least one Staphylococcus aureus (SA) antigen. Such a Staphylococcusaureus-MAPS (SA-MAPS) composition as disclosed herein is useful for theproduction of immunogenic compositions, such as those useful invaccines, as well as for treatment.

In some embodiments, the SA-MAPS immunogenic composition as disclosedherein generates an immune response in a subject, preferably an antibodyresponse and a B-cell and/or T-cell response. In some embodiments, theSA-MAPS immunogenic composition as disclosed herein generates a CD8+T-cell response, a CD4+ T-cell response or a CD8+/CD4+ T-cell response.The inventors demonstrate that mice immunized with or administered aSA-MAPS immunogenic composition as disclosed responded to SA antigensand produced significant amount of IFN-γ, IL-17A and IL-22,demonstrating that the SA-MAPS composition can generate of Th1, Th2,Th17 and Th22 responses. Accordingly, in some embodiments, a SA-MAPSimmunogenic composition as disclosed herein generates a T-cell responseand, more specifically, any one or more of a Th1, Th2, Th17 and Th22response to a SA peptide or protein present in the SA-MAPS composition.In some embodiments, a SA-MAPS immunogenic composition as disclosedherein generates an anti-polysaccharide antibody response and/or aB-cell and/or T-cell, e.g., Th1/Th2/Th17/Th22 response. In someembodiments, the immune response elicited by the SA-MAPS immunogeniccomposition as disclosed herein is an antibody or B cell response to atleast one antigenic polysaccharide, and an antibody or B cell responseand a CD4+ and/or CD8+ T cell response, including Th1, Th2, Th17 or Th22responses, or a CD8+ T cell response.

In some embodiments, a SA-MAPS immunogenic composition as disclosedherein elicits an immune response that results in activation of INF-γ,IL-17A, IL-17F, IL-21 or IL-22 producing cells, or produces INF-γ,IL-17A and IL-22 producing cells. This is important in that the SA-MAPSimmunogenic composition presents a major advantage by eliciting twoforms of immunity—that is, a conventional humoral (B-cell dependent)immune response to an immunogenic polysaccharide and SA-antigens, aswell as a T-cell response and, more specifically, any one or more ofTh17, Th1, Th2 or Th22 responses to a SA peptide or protein present inthe SA-MAPS composition. Moreover, in some embodiments, the SA-MAPSimmunogenic composition as disclosed herein can enhance specific B-cellor T-cell responses by modifying the protein/polysaccharide ratio,complex size, or by incorporating specific co-stimulatory factor, suchas TLR2/4 ligands, etc., into the composition.

In particular, the present invention is relates to compositionscomprising an immunogenic polysaccharide, at least one Staphylococcusaureus protein or peptide antigen; and at least one complementaryaffinity-molecule pair comprising (i) a first affinity molecule thatassociates with the immunogenic polysaccharide, and (ii) a complementaryaffinity molecule that associates with the Staphylococcus aureus proteinor peptide antigen, such that the first and complementary affinitymolecules serve as an indirect link between the immunogenicpolysaccharide and SA protein or peptide antigens. Such a system allowsfor a modular immunogenic composition, where one or more SA protein orpeptide antigens can be attached to the immunogenic polysaccharide in amodular fashion, allowing for flexibility in the number and type of SAantigens attached to immunogenic polysaccharide. Accordingly, theimmunogenic polysaccharide can attach at least 1, or at least 2, or aplurality of the same, or different SA protein or peptide antigens. Insome embodiments, the immunogenic polysaccharide is antigenic, and insome embodiments, the immunogenic polysaccharide is Type 5 (CP5) or Type8 (CP8), or a combination of Type 5 or Type 8 capsular polysaccharidefrom Staphylococcus aureus, or can be a pneumococcal capsularpolysaccharide, e.g., Type 1 (CP1) capsular polysaccharide from S.pneumoniae.

Staphylococcus aureus (SA) is a major cause of morbidity and mortalityworldwide. Vaccine development against SA has been challenging, likelydue to the complexity of pathogenesis and an incomplete understanding ofprotective immune mechanisms. The inventors previously developed avaccine platform referred to the Multiple-Antigen-Presenting-System(MAPS), as disclosed in US patent Application 2014/0154287, which isincorporated herein in its entirety by reference, which enables theinduction of broad adaptive immune responses. Herein, the inventors havedeveloped and optimized the system for the treatment and prevention ofinfection from Staphylococcus aureus. Herein, the inventors have used aSA-specific MAPS immunogenic composition which comprises 6 different SApeptide antigens to demonstrate that B- and T-cell mediated immunemechanisms contribute differentially to host defense against SA inmodels of skin necrosis, skin abscess, invasive disease or mucosalcolonization. In particular, immunization with a conventional subunitvaccine (i.e., a mixture of individual SA antigens not attached to ascaffold or polysaccharide), which induces solely humoral responses, orpassive transfer of rabbit anti-SA sera protected mice against sepsisand dermonecrosis infection, but had no impact on skin abscess infectionor gastrointestinal colonization by SA, against which antigen-specificT-cell immunity was both necessary and sufficient for protection. T-cellimmunity also contributed to protection in the sepsis and dermonecrosismodels, particularly when combined with antibody responses. Takentogether, the inventors have demonstrated that both humoral and cellularimmunity are important for host defense against SA. Herein, theinventors have demonstrated a SA-MAPS immunogenic composition as a novelvaccine to elicit multipronged adaptive responses, and is highlyvaluable in the development of effective and broadly protective vaccinesagainst SA.

In some embodiments, the SA-MAPS comprises at least one or more SAantigens, where the SA antigen is a antigenic protein or polypeptideselected from any of the group of: hemolysin (HI) (e.g., hemolysin α orHla), Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), Iron regulator surface proteinA (IsdA) and Iron regulator surface protein B (IsdB). In someembodiments, the SA-MAPS immunogenic composition as disclosed hereincomprises one or more peptide or polypeptide fragments of theseproteins, as long as the fragment is antigenic, and/or comprises one ormore epitopes to induce an immune response. Exemplary fragments can be,for example, but are not limited to Hla209(27-319), ClfA (221-559), ClfB(203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447). In someembodiments, a SA-MAPS immunogenic composition as disclosed hereincomprises at least 2, or at least 3, or at least 4, or at least 5, orall 6 peptide or polypeptide SA-antigens of Hla209(27-319), ClfA(221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447),or proteins or peptides of at least 85% sequence identity thereto. Insome embodiments, any of the above listed SA antigens can be substitutedfor a different SA peptide or polypeptide antigen known to one ofordinary skill in the art. Exemplary SA antigens can be any peptide orpolypeptide comprising at least part of the serine-aspirate repeatprotein E (SdrE) protein, Leukotoxin D (LukD) protein, or Leukotoxin E(LukE) protein, provided that the any peptide or polypeptide isimmunogenic, or is antigenic. Other SA antigens can be used, and aredisclosed herein.

The SA-MAPS immunogenic composition as disclosed herein can elicit bothhumoral and cellular responses to one or multiple SA antigens at thesame time. The SA-MAPS immunogenic compositions provide for along-lasting memory response, potentially protecting a subject fromfuture infection. This allows for a single SA-MAPS immunogeniccomposition that raise a high titer of functional anti-SA polysaccharideantibodies, and is similar or compares favorably with the antibody levelinduced by conventional conjugate vaccine. Moreover, there is norestriction to specific immunogenic polysaccharide used in the MAPSconstruct, which is typically a SA capsular polysaccharide or otherbacterial capsular or noncapsular polysaccharide, or the various SAantigen peptide or polypeptides used in SA-MAPS conjugate to generate arobust anti-polysaccharide antibody response. Additionally, the strongantibody response as well as Th17/Th1 and/or Th22 responses are specificto multiple SA protein antigens presented via the SA-MAPS composition.This is important in that the SA-MAPS immunogenic composition presents amajor advantage by eliciting two forms of immunity—that is, aconventional immune response to an immunogenic polysaccharide andSA-antigens, as well as a T-cell response and, more specifically, anyone or more of Th17, Th1, Th2 or Th22 responses to a SA peptide orprotein present in the SA-MAPS composition. Moreover, the SA-MAPSimmunogenic composition as disclosed herein provides a potential toenhance specific B-cell or T-cell responses by modifying theprotein/polysaccharide ratio, complex size, or by incorporating specificco-stimulatory factor, such as TLR2/4 ligands, etc., into thecomposition.

Accordingly, the SA-MAPS immunogenic composition as disclosed hereinuses an affinity-pair method to conjugate the SA antigens to theimmunogenic polysaccharide, therefore enabling a modular approach thatis easy and highly flexible for the preparation of a Staphylococcusaureus vaccine composition. The SA-MAPS immunogenic composition ishighly specific and stable; it can remain in the cold for months andretain its potency. The assembly process is simple enough to ensure highreproducibility; there are only a few steps required, which reduces therisk of lot-to-lot variation, of great industrial advantage. The SA-MAPSimmunogenic composition assembly is highly efficient (over 95%), even atlow concentrations of protein and polysaccharide (such as 0.1 mg/ml);this is a major advantage, because inefficiencies in conjugatemanufacture (typically efficiencies are in the <50% range) represent amajor hurdle and reason for the high cost of vaccines. For formulation:it is easy to adjust the composition and physical properties of thefinal product. The protein: polysaccharide ratio in the complex isadjustable; with moderate biotinylation of polymer, protein:polysaccharide can be 10:1 (w/w) or more; conversely, the ratio can be1:10 or less if such is the interest based on immunological goals.Additionally, the size of the immunogenic MAPS composition can beadjusted by the choice of immunogenic polysaccharide size. The methodsof making the SA-MAPS provide for ease in combining SA protein antigensand immunogenic polysaccharide with little modification, and allows thegeneration of a multivalent SA-MAPS composition by loading multiple SApeptide or protein antigens onto single immunogenic construct. As such,the SA-MAPS immunogenic composition as disclosed herein can be used todecrease the number of vaccines required to immunize a subject againstStaphylococcus aureus, in particular, different strains ofStaphylococcus aureus.

In some embodiments, the SA-MAPS immunogenic compositions as disclosedherein can be used to protect or treat a human susceptible to S. aureusinfection, by means of administering the immunogenic compositions via asystemic, dermal or mucosal route or be used to generate a polyclonal ormonoclonal antibody preparation that could be used to confer passiveimmunity on another subject. These administrations can include injectionvia the intramuscular, intraperitoneal, intradermal or subcutaneousroutes; or via mucosal administration to the oral/alimentary,respiratory or genitourinary tracts. In one embodiment, intranasaladministration is used for the treatment or prevention of nasopharyngealcarriage of S. aureus, thus attenuating infection at its earliest stage.In some embodiments, the SA-MAPS immunogenic compositions as disclosedherein may also be used to generate antibodies that are functional asmeasured by the killing of bacteria in either an animal efficacy modelor via an opsonophagocytic killing assay.

In some embodiments, aspects of the invention disclosed herein relate toa SA-MAPS immunogenic composition comprising an immunogenicpolysaccharide, at least one S. aureus peptide or polypeptide antigen,and at least one complementary affinity-molecule pair comprising: (a) afirst affinity molecule associated with the immunogenic polysaccharide,and (b) a complementary affinity molecule associated with the at leastS. aureus peptide or polypeptide antigen, where the first affinitymolecule associates with the complementary affinity molecule to link theS. aureus peptide or polypeptide antigen and the immunogenicpolysaccharide.

In some embodiments, the S. aureus peptide or polypeptide antigen isselected from any one or a combination of SA antigens selected from:hemolysin (Hl), Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), serine-aspirate repeat proteinE (SdrE), Iron regulator surface protein A (IsdA), Iron regulatorsurface protein B (IsdB), Leukotoxin D (LukD), and/or Leukotoxin E(LukE). In some embodiments, the SA-MAPS composition comprises ahemolysin (HI) S. aureus antigen and at least one additional S. aureusantigen selected from: Clumping factor A (ClfA), Clumping factor B(ClfB), serine-aspirate repeat protein D (SdrD), serine-aspirate repeatprotein E (SdrE), Iron regulator surface protein A (IsdA), Ironregulator surface protein B (IsdB), Leukotoxin D (LukD), or Leukotoxin E(LukE).

In some embodiments, the SA-MAPS composition comprises a hemolysin (HI)S. aureus antigen and at least two, or at least 3, or at least 4, or atleast 5 or more additional S. aureus antigen selected from any of thegroup comprising: Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), serine-aspirate repeat proteinE (SdrE), Iron regulator surface protein A (IsdA), Iron regulatorsurface protein B (IsdB), Leukotoxin D (LukD), or Leukotoxin E (LukE).In some embodiments, the SA-MAPS composition comprises a hemolysin α(Hla) antigen, and a Clumping factor A (ClfA) antigen, and a Clumpingfactor B (ClfB) antigen, and a serine-aspirate repeat protein D (SdrD)antigen, and a Iron regulator surface protein A (IsdA) antigen, and anIron regulator surface protein B (IsdB) antigen.

In some embodiments, the SA-MAPS composition comprises S. aureusantigens Hla209(27-319), ClfA (221-559), ClfB (203-542), SdrD (246-682),IsdA (47-324) and IsdB (48-447).

In some embodiments, the SA-MAPS composition comprises a Hl antigen is aα-hemolysin (Hla), a β-hemolysin (Hlb) or a γ-hemolysin (Hl-gamma) fromS. aureus, for example, a wildtype Hla (WT Hla) SA antigen or a Hla SAantigen with a reduced hemolytic activity or is a non-hemolytic Hlaprotein. In some embodiments, the SA-MAPS composition comprises a Hlaantigen with a reduced hemolytic activity comprises amino acids of SEQID NO: 14 (i.e., wt-Hla), SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18or a polypeptide with at least 85% sequence identity thereto. In someembodiments, a SA-MAPS composition as disclosed herein comprises a Hlaantigen with a reduced hemolytic activity that has amino acids of SEQ IDNO: 16 or a polypeptide with at least 85% sequence identity thereto.(i.e., Hla209 (27-319).

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa ClfA antigen comprises at least SEQ ID NO: 3 or a polypeptide with atleast 85% sequence identity to SEQ ID NO: 3 (i.e., aa 221-559 of ClfA).In some embodiments, a SA-MAPS composition as disclosed herein comprisesa ClfA antigen that is a fragment of at least 30 amino acids of SEQ IDNO: 2 or a polypeptide of at least 30 amino acids that has at least 85%sequence identity to a portion of SEQ ID NO: 2. (i.e., ClfA (40-899))

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa ClfB antigen which comprises at least SEQ ID NO: 5 or a polypeptidewith at least 85% sequence identity to SEQ ID NO: 5 (i.e., ClfB(203-542)). In some embodiments, a SA-MAPS composition as disclosedherein comprises a ClfB antigen that is a fragment of at least 30 aminoacids of SEQ ID NO: 4 or a polypeptide of at least 30 amino acids thathas at least 85% sequence identity to a portion of SEQ ID NO: 4 (i.e., afragment of ClfB protein).

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa SdrD antigen which comprises at least SEQ ID NO: 7 or a polypeptidewith at least 85% sequence identity to SEQ ID NO: 7 (i.e., SdrD(246-682)). In some embodiments, a SA-MAPS composition as disclosedherein comprises a SdrD antigen which is a fragment of at least 30 aminoacids of SEQ ID NO:6 or a polypeptide of at least 30 amino acids thathas at least 85% sequence identity to a portion of SEQ ID NO: 6. (i.e.,a fragment of aa 31-1281 (mature) of SdrD protein).

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa SdrE SA antigen which comprises a fragment of at least 30 amino acidsof SEQ ID NO:8 or a polypeptide of at least 30 amino acids that has atleast 85% sequence identity to a portion of SEQ ID NO: 8 (i.e., afragment ofs maure protein of SdrE)

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa IsdA SA-antigen which comprises at least SEQ ID NO: 11 or apolypeptide with at least 85% sequence identity to SEQ ID NO: 11 (i.e.,IsdA (47-324)). In some embodiments, a SA-MAPS composition as disclosedherein comprises a IsdA SA antigen which comprises a fragment of atleast 30 amino acids of SEQ ID NO:10 or a polypeptide of at least 30amino acids that has at least 85% sequence identity to a portion of SEQID NO: 10 (i.e., a fragment of aa 47-316 (mature) IsdA protein).

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa IsdB SA-antigen which comprises at least SEQ ID NO: 13 or apolypeptide with at least 85% sequence identity to SEQ ID NO: 13 (i.e.,IsdB (48-477). In some embodiments, a SA-MAPS composition as disclosedherein comprises a IsdB SA antigen which comprises a fragment of atleast 30 amino acids of SEQ ID NO:12 or a polypeptide of at least 30amino acids that has at least 85% sequence identity to a portion of SEQID NO: 12 (i.e., a fragment of aa 47-613 (mature) IsdB protein).

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa first affinity molecule which is biotin or a derivative or mimicmolecule thereof, for example, but not limited to, a biotin derivative,lipoic acid, HABA (hydroxyazobenzene-benzoic acid) or/and dimethyl-HABAor an amine-PEG3-biotin ((+)-biotinylation-3-6, 9-trixaundecanediamine).

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa complementary affinity molecule which is a biotin-binding protein, oran avidin-like protein, for example, but not limited to, any one or acombination of rhizavidin, avidin, streptavidin, or a homologue orderivative thereof. In some embodiments, a SA-MAPS composition asdisclosed herein comprises a complementary affinity molecule which isrhizavidin, and comprises amino acids of SEQ ID NO: 1, or 85% sequenceidentity to amino acids of SEQ ID NO: 1.

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa S. aureus antigen as a fusion protein comprising the S. aureus antigenfused to a complementary affinity binding molecule. In alternativeembodiments, the first affinity molecule is cross-linked to theimmunogenic polysaccharide.

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa first affinity molecule is cross-linked to the immunogenicpolysaccharide using a cross-linking reagent selected from any in thegroup consisting of: CDAP (1-cyano-4-dimethylaminopyridiniumtetrafluoroborate); EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride); sodium cyanoborohydride; cyanogen bromide; and ammoniumbicarbonate/iodoacetic acid. In some embodiments, the first affinitymolecule is cross-linked to carboxyl, hydroxyl, amino, phenoxyl,hemiacetal, and mecapto functional groups of the immunogenicpolysaccharide. In some embodiments, the first affinity molecule iscovalently bonded to the immunogenic polysaccharide.

In some embodiments, a SA-MAPS composition as disclosed herein comprisesa first affinity molecule and complementary affinity molecule pair,which can be selected from a group consisting of: biotin/biotin-bindingprotein, antibody/antigen, enzyme/substrate, receptor/ligand,metal/metal-binding protein, carbohydrate/carbohydrate binding protein,lipid/lipid-binding protein, His tag/His tag-binding substance. In someembodiments, wherein the antigen is non-covalently attached, orcovalently attached to the complementary affinity molecule.

In some embodiments, a secretion signal peptide is located at theN-terminal of the avidin-like protein, e.g., but not limited to asecretion signal sequence that comprises at least MKKIWLALAGLVLAFSASA(SEQ ID NO: 23) or MKKIWLALAGLVLAFSASAAQDP (SEQ ID NO: 24) or an aminoacid sequence having at least 85% identity thereof. In some embodiments,a flexible linker peptide is also attached to the antigen, wherein theflexible linker peptide attaches the antigen to the complementaryaffinity molecule.

In some embodiments, a SA-MAPS composition as disclosed herein comprisesan immunogenic polysaccharide is purified from living organisms or is asynthetic immunogenic polysaccharide, for example, where the livingorganism is selected from the group consisting of: bacteria, archaea,eukaryotic cells, fungi, insects, plants, animals, or chimeras thereof.

In some embodiments, a SA-MAPS composition as disclosed herein comprisesat least 3 S. aureus peptide or polypeptide antigens, or at least 5 S.aureus peptide or polypeptide antigens, or between 2-10 S. aureuspeptide or polypeptide antigens, or between 10-15 S. aureus peptide orpolypeptide antigens, or between 15-20 S. aureus peptide or polypeptideantigens, or between 20-50 S. aureus peptide or polypeptide antigens, orbetween 50-100 S. aureus peptide or polypeptide antigens, or more than100 S. aureus peptide or polypeptide antigens.

In some embodiments, a SA-MAPS composition as disclosed herein comprisesan immunogenic polysaccharide is selected from a polysaccharide from thegroup consisting of: S. aureus, Vi polysaccharide, pneumococcal capsularpolysaccharides, pneumococcal cell wall polysaccharide, Haemophilusinfluenzae Type b polysaccharide, Meningococcal polysaccharide,0-antigens from Gram-negative bacteria and other bacterial capsular orcell wall polysaccharides. In some embodiments, a SA-MAPS composition asdisclosed herein comprises an immunogenic polysaccharide selected fromtype 1 capsular polysaccharide (CP1) of Streptococcus pneumoniae, type 5capsular polysaccharide (CP5) of S. aureus or type 8 capsularpolysaccharide (CP8) of S. aureus.

In some embodiments, a SA-MAPS composition as disclosed herein, furthercomprises at least one co-stimulation factor associated to theimmunogenic polysaccharide, e.g., a co-stimulation factor is selectedfrom the group consisting of: Toll like receptor ligand or agonists, NODligand or agonists, or activator/agonists of the inflammasome. In someembodiments, the co-stimulation factor is attached to immunogenicpolysaccharide directly, or via a complementary affinity-molecule paircomprising: a first affinity molecule which associates with theimmunogenic polysaccharide, and a complementary affinity molecule whichassociates with the co-stimulation factor, wherein the first affinitymolecule associates with the complementary affinity molecule to link theco-stimulatory factor to the immunogenic polysaccharide.

In some embodiments, a SA-MAPS composition as disclosed herein is usedto elicit an immune response to S. aureus in a subject, for example,where the immune response is any of or a combination of: (i) an antibodyor B-cell response, (ii) an antibody or B-cell response and T-cellresponse, (iii) an immune response to at least one immunogenicpolysaccharide and at least one peptide or polypeptide S. aureusantigen, (iv) a CD4+ T cell response, including Th1, Th2, or Th17 orTh22 response, or a CD8+ T cell response, or CD4+ and CD8+ T cellresponse, (v) an antibody or B cell response to at least one antigenicpolysaccharide and a CD4+ T cell response, including Th1, Th2, or Th7 orTh22 response, or a CD8+ T cell response, or CD4+/CD8+ T cell responseto at least one peptide or polypeptide antigen, (vi) an antibody or Bcell response to at least one antigenic polysaccharide, and an antibodyor B cell response and a CD4+ T cell response, including Th1, Th2, Th7or Th22 responses, or a CD8+ T cell response, or CD4+/CD8+ T cellresponse to at least one peptide or polypeptide antigen, (vii) resultsin activation of INF-γ, IL-17A or IL-22 producing cells, or INF-γ,IL-17A and IL-22 producing cells, (viii) an antibody or B-cell responseagainst the S. aureus antigen which associates with the immunogenicpolysaccharide.

In some embodiments, a SA-MAPS composition as disclosed herein, furthercomprises at least one adjuvant.

In some embodiments, a SA-MAPS composition as disclosed herein is usedin a diagnostic for exposure to a pathogen or immune threat. In someembodiments, a SA-MAPS composition as disclosed herein is used inpreventing infection by S. aureus. In some embodiments, a SA-MAPScomposition as disclosed herein is used for preventing colonization of asubject by S. aureus.

Another aspect of the technology disclosed herein relates to a methodfor inducing an immune response in a subject to S. aureus, comprisingadministering to the subject a SA-MAPS composition as disclosed herein.For example, the SA-MAPS composition as disclosed herein is used toinduce an immune response in a subject to S. aureus, where the immuneresponse is, for example, any of or a combination of: (i) an antibody orB-cell response, (ii) an antibody or B-cell response and T-cellresponse, (iii) an immune response to at least one immunogenicpolysaccharide and at least one peptide or polypeptide S. aureusantigen, (iv) a CD4+ T cell response, including Th1, Th2, or Th17 orTh22 response, or a CD8+ T cell response, or CD4+ and CD8+ T cellresponse, (v) an antibody or B cell response to at least one antigenicpolysaccharide and a CD4+ T cell response, including Th1, Th2, or Th7 orTh22 response, or a CD8+ T cell response, or CD4+/CD8+ T cell responseto at least one peptide or polypeptide antigen, (vi) an antibody or Bcell response to at least one antigenic polysaccharide, and an antibodyor B cell response and a CD4+ T cell response, including Th1, Th2, Th17or Th22 responses, or a CD8+ T cell response, or CD4+/CD8+ T cellresponse to at least one peptide or polypeptide antigen, (vii) resultsin activation of INF-γ, IL-17A or IL-22 producing cells, or INF-γ,IL-17A and IL-22 producing cells, (viii) an antibody or B-cell responseagainst the S. aureus antigen which associates with the immunogenicpolysaccharide.

Another aspect of the technology disclosed herein relates to a method ofvaccinating a mammal against at least one antigen-bearing pathogen, themethod comprising administering to the mammal a SA-MAPS composition asdisclosed herein. In some embodiments, the subject or mammal is a human.In alternative embodiments, the subject or mammal is an agricultural ornon-domestic animal, or a domestic animal.

In some embodiments, a SA-MAPS composition as disclosed herein isadministered via subcutaneous, intranasal, intradermal, or intramuscular injection, or via transdermal skin patch.

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and at least one S. aureuspeptide or polypeptide antigen, wherein the rhizavidin protein comprisesamino acids of SEQ ID NO: 1, or 85% sequence identity to amino acids ofSEQ ID NO: 1, and the S. aureus peptide or polypeptide comprises afragment of at least 20 amino acids of a protein selected from any of:haemolysin (HI), Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), serine-aspirate repeat proteinE (SdrE), Iron regulator surface protein A (IsdA), Iron regulatorsurface protein B (IsdB), Leukoptoxin D (LukD), or Leukoptoxin E (LukE).In some embodiments, a fusion protein as disclosed herein comprises a S.aureus peptide selected from any of Hla209(27-319), ClfA (221-559), ClfB(203-542), SdrD (246-682), IsdA (47-324) and IsdB (48-447).

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises anon-hemolytic variant of a Hla protein (i.e. Rhavi-Hla209). In someembodiments, a non-haemolytic variant of a Hla protein comprises atleast SEQ ID NO: 16 or a protein of at least 20 amino acids that has atleast 85% sequence identity to SEQ ID NO: 16. (i.e., Hla209(aa 27-319).

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises a fragment ofat least 20 amino acids of a Clumping factor A (ClfA) protein (i.e.Rhavi-ClfA). In some embodiments, a ClfA protein comprises at least SEQID NO: 3 or a protein of at least 20 amino acids that has at least 85%sequence identity to SEQ ID NO: 3 (i.e., ClfA (221-559)).

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises a fragment ofat least 20 amino acids of a Clumping factor B (ClfB) protein (i.e.,Rhavi-ClfB). In some embodiments, a ClfB protein comprises at least SEQID NO: 5 or a protein of at least 20 amino acids that has at least 85%sequence identity to SEQ ID NO: 5. (i.e., ClfB (203-542)).

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises a fragment ofat least 20 amino acids of a serine-aspirate repeat protein D (SdrD)protein (i.e. Rhavi-SdrD). In some embodiments, a SdrD protein comprisesat least SEQ ID NO: 7 or a protein of at least 20 amino acids that hasat least 85% sequence identity to SEQ ID NO: 7 (i.e., SdrD (246-682)).

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises a fragment ofat least 20 amino acids of a serine-aspirate repeat protein D (SdrE)protein (i.e. Rhavi-SdrE). In some embodiments, a SdrE protein comprisesat least SEQ ID NO: 8 or a protein of at least 20 amino acids that hasat least 85% sequence identity to SEQ ID NO: 8.

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises a fragment ofat least 20 amino acids of Iron regulator surface protein A (IsdA),protein (i.e. Rhavi-IsdA). In some embodiments, a IsdA protein comprisesat least SEQ ID NO: 11 or a protein of at least 20 amino acids that hasat least 85% sequence identity to SEQ ID NO: 11 (i.e., IsdA (47-324).

Another aspect of the technology disclosed herein relates to a fusionprotein comprising a rhizavidin protein and an S. aureus peptide orpolypeptide antigen, wherein the rhizavidin protein comprises aminoacids of SEQ ID NO: 1, or 85% sequence identity to amino acids of SEQ IDNO: 1, and the S. aureus peptide or polypeptide comprises a fragment ofat least 20 amino acids of Iron regulator surface protein B (IsdB),protein (i.e. Rhavi-IsdB). In some embodiments, a IsdB protein comprisesat least SEQ ID NO: 13 or a protein of at least 20 amino acids that hasat least 85% sequence identity to SEQ ID NO: 13 (i.e., IsdB (48-477)).

Another aspect of the technology disclosed herein relates to a kitcomprising: (a) a container comprising an immunogenic polysaccharidecross-linked with a plurality of first affinity molecules; and (b) acontainer comprising a complementary affinity molecule which associateswith the first affinity molecule, wherein the complementary affinitymolecule associates with at least one S. aureus antigen. In someembodiments, a kit can further comprise any one or more of: (i) a meansor agent to attach the complementary affinity molecule to the antigen,(ii) at least one co-stimulation factor, (iii) a cross-linking reagentwhich can be selected from the group consisting of: CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC(1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride), sodiumcyanoborohydride, cyanogen bromide, or ammonium bicarbonate/iodoaceticacid for linking the co-factor to the polysaccharide, (iv) a containercomprising an expression vector for expressing an antigen-affinitymolecule fusion protein, for example, an expression vector that canoptionally comprise a sequence for a linker peptide, wherein theexpression vector can expresses an antigen-affinity molecule fusionprotein comprising a linker peptide between the antigen and the affinitymolecule, and/or (v) one or more of a fusion protein as disclosedherein, wherein the fusion protein is selected from any of: (i) a fusionprotein comprising a the C-terminal of SEQ ID NO: 1 (or a protein of atleast 80% or 85% or more sequence identity thereto) is fused to any ofhemolysin (HI) (e.g., hemolysin α or Hla209), Clumping factor A (ClfA),Clumping factor B (ClfB), serine-aspirate repeat protein D (SdrD), Ironregulator surface protein A (IsdA) and Iron regulator surface protein B(IsdB), or fragments thereof, or (ii) a fusion protein comprising a theC-terminal of SEQ ID NO: 1 (or a protein of at least 80% or 85% or moresequence identity thereto) fused to any one of: Hla209(27-319), ClfA(221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447)or proteins or peptides having at least 85% sequence identity thereto,or (iii) a fusion protein selected from any of Rhavi-HLA209-ClfA,Rhivi-HLA209-ClfB, Rhivi-HLA209-SdrD, Rhavi-HLA209-IsdA,Rhavi-HLA209-IsdB, Rhavi-ClfA-ClfB, Rhavi-ClfA-SdrD, Rhavi-ClfA-IsdA;Rhavi-ClfA-IsdB; Rhavi-ClfB-SdrD; Rhavi-ClfB-IsdA; Rhavi-ClfB-IsdB,Rhavi-SdrD-IsdA; Rhavi-SdrD-IsdB; Rhavi-IsdA-IsdB, where CLFA=CLFAprotein or a fragment thereof, e.g., ClfA (221-559), CLFB=ClfB proteinor a fragment thereof, e.g., ClfB (203-542), SDRD=SdrD protein or afragment thereof, e.g., SdrD (246-682), ISDA=IsdA protein or a fragmentthereof, e.g., IsdA (47-324); ISDB=IsdB protein or a fragment thereof,e.g., IsdB (48-477); HLA209=Hla protein with the 209 mutation, or afragment thereof, e.g., Hla209(27-319).

Accordingly, one aspect of the present invention relates to animmunogenic composition comprising a polymer, at least one protein orpeptide antigen, and at least one complementary affinity-molecule pair,where the complementary affinity-molecule pair comprises a firstaffinity molecule that associates with the polymer and a complementaryaffinity molecule that associates with the protein or peptide antigen,so that when the first affinity molecule associates with thecomplementary affinity molecule, it indirectly links the antigen to thepolymer.

Provided herein also is a method of vaccinating a subject, e.g., amammal, e.g., a human with the immunogenic compositions as disclosedherein, the method comprising administering a vaccine compositioncomprising a SA-MAPS composition as disclosed herein to the subject.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the preparation of SA-Mix and SA-MAPS vaccines. FIG. 1Ashows hemolytic activity of wild type (WT) Hla, Hla209 mutant and theirrhizavidin (rhavi) fusions. One HU was defined as the activity thatcauses 50% hemolysis of 1% rabbit red blood cells in PBS (pH7.5) after30 min incubation at 37° C. and expressed per 1 mg/ml. Fusion withrhizavidin significantly reduced the hemolytic activity of WT Hla.Symbols represent mean±SEM. FIG. 1B shows schematics of SA-Mix andSA-MAPS vaccine. SA-Mix vaccine contains six S. aureus antigens mixed atequal molar ratio. SA-MAPS complex was prepared by coupling rhavi fusionantigens with a biotinylated pneumococcal type-1 capsular polysaccharide(SP PS1 or CP1). FIG. 1C shows results of SDS-PAGE of the purifiedSA-MAPS complex. MAPS complexes were treated with reducing-SDS samplebuffer at room temperature (RT) or at 100° C. (Boil) for 10 min beforeapplied to SDS-PAGE. The affinity coupling between rhavi-fusion antigensand biotinylated PS in MAPS complexes was retained after SDS-treatmentunless the sample was boiled.

FIGS. 2A and 2B show antigen-specific immune responses induced by SA-Mixor SA-MAPS vaccine. FIG. 2A is a histogram of antigen-specific IgG titerof C57BL/6 mice (n=10 per group) after three subcutaneous immunizationswith SA-Mix or SA-MAPS vaccine. Control group received adjuvant alone(Alum). Immunization of mice with SA-MAPS induced significantly highertiter of IgG antibody to each target antigen than what was induced bySA-Mix. FIG. 2B shows that IFNγ, IL-17A and IL22 production, indicativeof antigen-specific T-cell responses after SA-Mix or SA-MAPS. SA-MAPSbut not SA-Mix eliciting antigen-specific T-cell responses. a.u.,arbitrary unit. Bars represent Geometric means +/−95% CI. *, P<0.05; **,P<0.01; ***, P<0.001.

FIGS. 3A-3E show SA-MAPS confers enhanced and broad protection in modelsof S. aureus infection and colonization. Mice (n=10 per group) receivedthree immunizations with SA-Mix or SA-MAPS vaccine. Control groupreceived adjuvant alone (Alum). Mice were infected with S. aureus 3weeks after the last immunization. FIG. 3A (left) shows a Kaplan-Mahersurvival curve after vaccination with either SA-Mix or SA-MAPS, anddemonstrated that SA-MAPS protected animals from sepsis infection of S.aureus. Moreover, FIG. 3A (right) shows that mice that received SA-MAPSvaccine had delayed onset illness compared to the control group or thegroup that received SA-Mix. FIG. 3B and FIG. 3C shows area of lesionafter mice are vaccinated with SA-Mix or SA-MAPS, with SA-MAPS reducingthe incidence (FIG. 3C) and the extent (FIG. 3B) of dermonecrosis afterskin infection with S. aureus. FIG. 3B (left) shows area of lesion ofmice immunized with SA-MAPS (closed square), SA-Mix (closed triangle) ascompared to Alum controls (open square). Inset of FIG. 3B (left) is arepresentative picture of dermonecrotic lesion (black arrow) after S.aureus infection. FIG. 3B (right) is a histogram showing thatvaccination with SA-MAPS (closed squares) but not with SA-Mix (closedtriangles) protected against skin abscess formation caused by S. aureus.Symbols represent Mean surface area SEM. FIG. 3D shows the CFU ofbacteria recovered from skin abscesses on day 4 after infection. FIG. 3Eshows CFU with SA-Mix (closed triangles) or SA-MAPS (closed squares), ascompared to the control Alum group (open squares), and demonstrates thatsignificantly fewer animals that received SA-MAPS vaccine developed skinabscess after inoculation. FIG. 3E (inset) are representative picture ofskin abscess of SA-MAPS (top) or SA-Mix (bottom) (arrows). Barsindicated Geometric means. Dashed line indicated the detection limit(22.5 CFU).

FIGS. 4A-4C show that vaccination with SA-MAPS but not with SA-Mixfacilitates the clearance of GI colonization of SA. FIG. 4A shows CFUper gram of feces of C57BL/6 mice (n=5) inoculated intranasally with5×10⁷ CFU of USA300 LAC^(strep) strain. Fecal pellets were collected ondays 1 (D1), 4 (D4), 7 (D7) and 11 (D11) after inoculation. Stable SAcolonization is apparent between days 4 and 11 post-inoculation. FIGS.4B and 4C show that vaccination with SA-MAPS, but not with SA-Mix,significantly reduced the density of GI colonization of SA over time.FIG. 4B shows the CFU of bacteria recovered from fecal samples. FIG. 4Cshows the percentage of CFU on day-7 (D7) post inoculation as comparedto CFU on day-1 (D1) post inoculation. Bars represent Geometric means.Dashed lines indicated the detection limit (40 CFU). N.S, notsignificant; **, P<0.01.

FIGS. 5A-5D show roles of antigen-specific antibodies in the protectionagainst different types of & aureus infection and colonization. Mice(n=10 per group) received intraperitoneal injection with 200 μl ofheat-inactivated, pre- or post-SA-MAPS immunization rabbit sera one dayprior S. aureus infection. Infusion with rabbit sera against S. aureusantigens protected mice against sepsis (FIG. 5A) and dermonecrosisinfection (FIG. 5B), but had no impact on the formation of skin abscess(FIG. 5C) or the clearance of S. aureus from the GI tracts (FIG. 5D).Symbols in B represent Mean±SEM. Bars in C and D Geometric means. Dashedlines indicated the detection limit (22.5 CFU for abscess infection and40 CFU for GI colonization model). N.S, not significant; *, P<0.05; **,P<0.01; ***, P<0.001.

FIGS. 6A-6F show the contributions of antigen-specific T-cell responsesto protection against different types of & aureus infection andcolonization. FIG. 6A is a histogram of antigen-specific IgF and showsthat immunization of μMT^(−/−)- (B-cell deficient) mice with SA-MAPSelicited no IgG antibodies. FIG. 6B shows levels of IFNγ, IL-17 andIL-22 production after immunization of μMT^(−/−)- (B-cell deficient)mice with SA-MAPS, showing normal T-cell responses to S. aureusantigens. Bars represent Geometric mean +95% CI. FIG. 6C shows thegeneration of antigen-specific T cell immunity slightly delays onset ofillness, but does not provide significant protection. FIG. 6D showsantigen-specific T cell immunity provided partial protection againstdermonecrosis, especially in the early stage of the infection. Symbolsrepresent Mean±SEM. FIGS. 6E and 6F show that in the absence ofantibodies, SA MAPS provides protection against abscess formation duringskin infection (FIG. 6E) and accelerates clearance of GI colonization ofS. aureus (FIG. 6F). Bars represent Geometric means. Dashed linesindicated the detection limit (22.5 CFU for abscess infection and 40 CFUfor GI colonization model). N.S, not significant; *, P<0.05; ***,P<0.001.

FIG. 7 shows the contribution of antibodies using passive immunization.The antigen-specific IgG for ClfA, ClfB, IsdA, IsdB, SdrD or Hla afterimmunizing rabbits with SA-MAPS (P3, filled squares) as compared topre-vaccination rabbit sera (Pre, open squares) which were used ascontrols.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates immunogenic compositions and compositionscomprising an immunogenic complex that comprises at least oneStaphylococcus aureus antigen, or multiple Staphylococcus aureusantigens, attached to an immunogenic polysaccharide scaffold for use ineliciting an immune response (both a cellular and humoral immuneresponse) to each of the SA antigens attached to the immunogenicpolysaccharide and to the immunogenic polysaccharide, when administeredto a subject.

More specifically, disclosed herein is an immunogenic Multiple AntigenPresenting System (MAPS) comprising an immunogenic polysaccharide, andattached to the immunogenic polysaccharide via an affinity binding pair,at least one Staphylococcus aureus (SA) antigen. Such a Staphylococcusaureus-MAPS (SA-MAPS) composition as disclosed herein is useful for theproduction of immunogenic compositions, such as those useful invaccines, as well as for treatment. The SA-MAPS immunogenic compositionas disclosed herein stimulates a humoral and cellular immune response:it can generate anti-polysaccharide antibody and the B-cell and T-cell,e.g., Th1/Th17 responses to multiple Staphylococcus aureus (SA) antigenusing single SA-MAPS immunogenic construct. A combination of B- andT-cell immunity to Staphylococcus aureus will be a useful vaccinestrategy against Staphylococcus aureus invasive diseases, as well asfrom mild skin infections to endocarditis, dermonecrosis, osteomyelitis,bacteremia, sepsis, and other forms of disease associated withStaphylococcus aureus.

The inventors previously developed a vaccine platform referred to theMultiple-Antigen-Presenting-System (MAPS), as disclosed in US patentApplication 2014/0154287, which is incorporated herein in its entiretyby reference, which enables the induction of broad adaptive immuneresponses. Herein, the inventors have developed and optimized the systemfor the treatment and prevention of infection from Staphylococcusaureus.

In particular, the inventors have generated a SA-MAPS immunogeniccomposition comprising an immunogenic polysaccharide (typically SA CP5,CP8 or S. pneumoniae CP1, or other PS or variants or combinationsthereof), at least one Staphylococcus aureus protein or peptide antigen;and at least one complementary affinity-molecule pair comprising (i) afirst affinity molecule that associates with the immunogenicpolysaccharide, and (ii) a complementary affinity molecule thatassociates with the Staphylococcus aureus protein or peptide antigen,such that the first and complementary affinity molecules serve as anindirect link between the immunogenic polysaccharide and SA protein orpeptide antigens. Such a system allows for a modular immunogeniccomposition, where one or more SA protein or peptide antigens can beattached to the immunogenic polysaccharide in a modular fashion,allowing for flexibility in the number and type of SA antigens attachedto immunogenic polysaccharide. Accordingly, the immunogenicpolysaccharide can attach at least 1, or at least 2, or a plurality ofthe same or different SA protein or peptide antigens. In someembodiments, the immunogenic polysaccharide is antigenic, and in someembodiments, the immunogenic polysaccharide is Type 5 (CP5) or Type 8(CP8), or a combination of Type 5 or Type 8 capsular polysaccharide fromStaphylococcus aureus, or can be a pneumococcal capsular polysaccharide,e.g., Type 1 (CP1) capsular polysaccharide from S. pneumoniae.

Herein, the inventors have used a SA-specific MAPS immunogeniccomposition which comprises 6 different SA peptide antigens todemonstrate that B- and T-cell mediated immune mechanisms contributedifferentially to host defense against SA in models of skin necrosis,skin abscess, invasive disease or mucosal colonization.

In some embodiments, the SA-MAPS comprises at least one or more SAantigens, where the SA antigen is an antigenic protein or polypeptideselected from any of the group of: hemolysin (HI) (e.g., hemolysin α orHla), Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), Iron regulator surface proteinA (IsdA) and Iron regulator surface protein B (IsdB). In someembodiments, the SA-MAPS immunogenic composition as disclosed hereincomprises one or more peptide or polypeptide fragments of theseproteins, as long as the fragment is antigenic, and/or comprises one ormore epitopes to induce an immune response. Exemplary fragments can be,for example, but are not limited to Hla209(27-319), ClfA (221-559), ClfB(203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447). In someembodiments, a SA-MAPS immunogenic composition as disclosed hereincomprises at least 2, or at least 3, or at least 4, or at least 5, orall 6 peptide or polypeptide SA-antigens of Hla209(27-319), ClfA(221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447),or proteins or peptides of at least 85% sequence identity thereto. Insome embodiments, any of the above listed SA antigens can be substitutedfor a different SA peptide or polypeptide antigen known to one ofordinary skill in the art. Exemplary SA antigens can be any peptide orpolypeptide comprising at least part of the serine-aspirate repeatprotein E (SdrE) protein, Leukotoxin D (LukD) protein, or Leukotoxin E(LukE) protein, provided that the any peptide or polypeptide isimmunogenic, or is antigenic. Other SA antigens can be used, and aredisclosed herein.

Accordingly, the embodiments herein provide for an immunogeniccomposition and methods useful for raising an immune response toStaphylococcus aureus in a subject, which can be used on its own or inconjunction or admixture with essentially any existing vaccineapproaches.

Staphylococcus aureus Multiple-Antigen Presenting System (SA-MAPS)

While it is envisioned that the SA-MAPS immunogenic composition asdisclosed herein comprises immunogenic polysaccharides fromStaphylococcus aureus, the SA-MAPS can use immunogenic polysaccharidesfrom a variety of different bacterial cells. In some embodiments, theimmunogenic polysaccharide is for example, but not limited to, Type 5(CP5) or Type 8 (CP8), or a combination of Type 5 or Type 8 capsularpolysaccharide from Staphylococcus aureus, or can be a pneumococcalcapsular polysaccharide, e.g., Type 1 (CP1) capsular polysaccharde fromS. pneumoniae or other capsular or noncaspular PS. In some embodiments,the polysaccharide is a capsular polysaccharide. In some embodiments,the polysaccharde is not a capsular polysaccharide (i.e., a noncapsularPS). With the different combinations of immunogenic polysaccharides anddifferent combinations of SA peptide or polypeptide antigens, theSA-MAPS composition is a flexible and versatile composition that can bedesigned and manufactured to elicit a particular, broad spectrum immuneresponse to Staphylococcus aureus. Table 1 provides a simple exampleguide for envisioning the flexibility of SA-MAPS embodiments.

Table 1 shows the versatility of the SA-MAPS platform: SA-MAPS comprisesan antigenic polysaccharide backbone and at least one SA-antigen, andoptionally one or more non-SA antigens. The antigenic or immunogenicpolysaccharide backbone can be a synthetic or antigenic polysaccharidefrom Staphylococcus aureus or alternatively a different a pathogen(exemplary antigenic polysaccharides are listed in the last column). ASA-MAPS composition can comprise at least one SA-antigen (exemplary SAantigens are listed), and can optionally comprise non-SA antigens.

TABLE 1 SA-MAPS Immunogenic polymer backbone:                 Antigens:Immunogenic polysaccharide         Other immunogenic polysaccharidesfrom viruses etc. SA-Antigens               Non-SA antigens synthetic    From pathogen               e.g., Hla209, ClfA, ClfB, SdrD, IsdA, IsdBetc.         Bacterial proteins/toxins Viral proteins Cancer antigensPlant toxins

Polysaccharides

One component of the SA-MAP immunogenic composition as disclosed hereinis a “backbone,” typically an antigenic or immunogenic polysaccharide(PS), and can comprise additional elements that do not negatively impactthe antigenic polysaccharide's function of (i) inducing an immuneresponse to the polysaccharide and (ii) presenting the associatedSA-antigen(s) to the immune system in immunogenic fashion. In someembodiments, the immunogenic polysaccharide is a syntheticpolysaccharide.

It is envisioned that the polysaccharide used in the SA-MAPS compositionis immunogenic, that is, it helps induce a specific immune response, andherein is referred to as an “immunogenic polysaccharide” or “antigenicpolysaccharide”. The specific immune response recognizes the particularimmunogenic PS and provides a unique response to the immunogenic complexas opposed to a different immunogenic complex. As explained herein, theresponse includes both a humoral and cell-mediated response.

In some embodiments, the immunogenic polysaccharide is a naturallyoccurring polysaccharide, e.g., a polysaccharide derived or purifiedfrom bacterial cells, and can be, for example, a capsular or noncaspularPS. In some embodiments, the immunogenic polysaccharide is derived orpurified from eukaryotic cells, e.g., fungi, insect or plant cells. Inyet other embodiments, the immunogenic polysaccharide is derived frommammalian cells, such as virus-infected cells or cancer cells. Ingeneral, such immunogenic polysaccharides are well known in the art andare encompassed for use in the methods and compositions as disclosedherein.

Staphylococcal microorganisms capable of causing invasive diseasegenerally also are capable of producing a capsule polysaccharide (CP)that encapsulates the bacterium and enhances its resistance to clearanceby the host innate immune system. The CP serves to cloak the bacterialcell in a protective capsule that renders the bacteria resistant tophagocytosis and intracellular killing. Bacteria lacking a capsule aremore susceptible to phagocytosis. Capsular polysaccharides arefrequently an important virulence factor for many bacterial pathogens,including Haemophilus influenzae, Streptococcus pneumoniae and Group Bstreptococci. In some embodiments, an immunogenic polysaccharide for usein the SA-MAPS immunogenic composition as disclosed herein is apolysaccharide or oligosaccharide from Gram-positive bacteria, forexample, a Staphylococcus aureus capsular polysaccharide.

Type 5 and Type 8 Polysaccharides from S. aureus

Most strains of S. aureus that cause infection in man contain eitherType 5 or Type 8 polysaccharides. Approximately 60% of human strains areType 8 and approximately 30% are Type 5. The structures of Type 5 andType 8 capsular polysaccharide antigens are described in Moreau et alCarbohydrate Res. 201; 285 (1990) and Fournier et al Infect. Immun. 45;87 (1984). Both have FucNAcp in their repeat unit as well as ManNAcAwhich can be used to introduce a sulfhydryl group.

Recently (Jones Carbohydrate Research 340, 1097-1106 (2005)) NMRspectroscopy revised the structures of the capsular polysaccharides to:

Type 5→4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→Type 8→3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→

Polysaccharides may be extracted from the appropriate strain of S.aureus using methods well known to the skilled man, for instance asdescribed in U.S. Pat. No. 6,294,177 or Infection and Immunity (1990)58(7); 2367, Fournier et al. (1984), supra; Fournier et al. (1987) Ann.Inst. Pasteur/Microbiol. 138:561-567; US Patent Application PublicationNo. 2007/0141077; and Int'l Patent Application Publication No. WO00/56357; each of which is incorporated herein by reference as if setforth in its entirety). For example, ATCC 12902 is a Type 5 S. aureusstrain and ATCC 12605 is a Type 8 S. aureus strain. In addition, theycan be produced using synthetic protocols. Moreover, serotype 5 or 8capsular polysaccharide can be recombinant produced using geneticengineering procedures also known to one of ordinary skill in the art(see, Sau et al. (1997) Microbiology 143:2395-2405; and U.S. Pat. No.6,027,925; each of which is incorporated herein by reference as if setforth in its entirety).

One S. aureus strain that can be used to obtain isolated serotype 8capsular polysaccharide is S. aureus R2 PFESA0286. This strain wasselected by flow cytometry with rabbit anti-serotype 8 polysaccharideantibodies after cultivation of S. aureus PFESA0286 (American TypeCulture Collection; Manassas, Va.: ATCC Accession No. 495:25) inModified Frantz Broth. Two populations, R1 and R2, were observed duringflow cytometry. R1 and R2 were purified and re-cultured. R2 yielded aserotype 8 capsular polysaccharide. Flow cytometric analysis showed ahomogenous fluorescence intensity. As such, R2 was selected for serotype8 capsular polysaccharide production.

One S. aureus strain that can be used to obtain isolated serotype 5capsular polysaccharide is S. aureus PFESA0266. This strain producesserotype 5 capsular polysaccharide during growth, and production peakswhen cells are in a stationary phase. Other S. aureus type 5 or type 8strains can be used to make the respective polysaccharides that areobtained either from established culture collections or clinicalspecimens.

In some embodiments, a Becker or Newman S. aureus strain can be used toobtain isolated serotype 5 capsular polysaccharide (CP5). In someembodiments, the Newman S. aureus strain can be used to obtain isolatedserotype 5 capsular polysaccharide (CP5).

In some embodiments, a Becker or Newman S. aureus strain can be used toobtain isolated serotype 8 capsular polysaccharide (CP8). In someembodiments, the Becker S. aureus strain can be used to obtain isolatedserotype 8 capsular polysaccharide (CP8).

Polysaccharides are of native size or alternatively may be sized, forinstance by microfluidisation, ultrasonic irradiation or by chemicaltreatment. The invention also covers oligosaccharides derived from thetype 5 and 8 polysaccharides from S. aureus.

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein can comprises a Type 5 (CP5), orType 8 (CP8) capsular polysaccharides (CP), or any of thepolysaccharides or oligosaccharides or lipopolysaccharides fromStaphylococcus aureus. In some embodiments, an immunogenicpolysaccharide for use in the SA-MAPS complex as disclosed herein cancomprises a capsular polysaccharide from a non-typeable (NT) SA strain,e.g., a cell wall surface antigen 336 (Type 336) or a polyribitolphosphate N-acetylglucosamine, which resembles cell wall teichoic acid.Type 336 isolates do not express capsule but do express cell surfacepolysaccharide or the 336 polysaccharide (336PS), which resembles S.aureus cell wall teichoic acid (Ma, J., et al., 2004. Evaluation ofserotypes of Staphylococcus aureus strains used in the production of abovine mastitis bacterin. J. Dairy. Sci. 87:178-182 14, 17; O'Brien, etal., 2000. Production of antibodies to Staphylococcus aureus serotypes5, 8, and 336 using poly(dl-lactide-co-glycolide) microspheres. J. DairySci. 83:1758-1766).

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein can comprises a capsularpolysaccharide (CP) from a methicillin-resistant S. aureus (MRSA),including hospital-acquired MRSA (HA-MRSA), or community-acquired MRSA(CA-MRSA) or any polysaccharides or oligosaccharides orlipopolysaccharides from MRSA, e.g., e.g., any one or more of a CP5, orCP8 from HA-MSSA and/or CA-MRSA. In alternative embodiments, animmunogenic polysaccharide for use in the SA-MAPS complex as disclosedherein can comprises a capsular polysaccharide (CP) from amethicillin-sensitive S. aureus (MSSA), e.g., any one or more of a CP5,or CP8 from MSSA.

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein can comprise more than one type ofpolysaccharide. For example, an immunogenic polysaccharide for use inthe SA-MAPS complex as disclosed herein can comprise a portion ofpolysaccharide A (e.g., Type 5 from SA), and the remaining portion ofpolysaccharide B (Type 8 from SA). The antigenic polysaccharide does notneed to be from the same organism, e.g., for example an immunogenicpolysaccharide for use in the SA-MAPS complex as disclosed herein cancomprise a portion of polysaccharide A (e.g., Type 5 or Type 8 from SA),and the remaining portion of polysaccharide B (e.g., a pneumococcuspolysaccharide or other bacterial capsular PS or noncapsular PS). Thereis no limit to the amount of different types of immunogenicpolysaccharides which can be used in a single MAPS backbone entity. Insome embodiments, where the immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein is a branched polymer, the chainpolysaccharide can be polysaccharide A, and the branches can be at least1 or at least 2 or at least 3 or more different antigenicpolysaccharides.

In some embodiments, the immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein is a branched polymer. In someembodiments, an immunogenic polysaccharide for use in the SA-MAPScomplex as disclosed herein is a single chain polymer.

In some embodiments, the immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein comprises at least 10 carbohydraterepeating units, or at least 20, or at least 50, or at least 75, or atleast 100, or at least 150, or at least 200, or at least 250, or atleast 300, or at least 350, or at least 400, or at least 450, or atleast 500, or more than 500 repeating units, inclusive.

In one aspect of the invention, the immunogenic polysaccharide (PS) foruse in the SA-MAPS complex as disclosed herein can have a molecular massof <500 kDa or >500 kDa. In another aspect of the invention, the PS hasa molecular mass of <70 kDa. In some embodiments, an immunogenicpolysaccharide for use in the SA-MAPS complex as disclosed herein is alarge molecular weight polymer, e.g., a polymer can be of an averagemolecular weight of between about 425-500 kDa, inclusive, for example,at least 300 kDa, or at least 350 kDa, or at least 400 kDa, or at least425 kDa, or at least 450 kDa, or at least 500 kDa or greater than 500kDa, inclusive, but typically less than 500 kDa. In some embodiments, animmunogenic polysaccharide for use in the SA-MAPS complex as disclosedherein can be a small molecular weight polymer, e.g., a polymer can beof an average molecular weight of between about 60 kDA to about 90 kDa,for example, at least 50 kDa, or at least 60 kDa, or at least 70 kDa, orat least 80 kDa, or at least 90 kDa, or at least 100 kDa, or greaterthan 100 kDa, inclusive, but generally less than about 120 kDa.

In some embodiments, the immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein is harvested and purified from anatural source; and in other embodiments, the polysaccharide issynthetic. Methods to produce synthetic polymers, including syntheticpolysaccharides, are known to persons of ordinary skill and areencompassed in the compositions and methods as disclosed herein.

In some embodiments, a type 5 and/or type 8 capsular polysaccharide oroligosaccharide included in a SA-MAPS immunogenic compositions asdisclosed herein has a molecular weight of between 20 kDa and 1000 kDa.In some embodiments, the type 5 and/or type 8 and/or type 1 capsularpolysaccharide or oligosaccharide of a SA-MAPS immunogenic compositionsas disclosed herein has a molecular weight of between 200 kDa and 5000kDa, or a molecular weight range of between 70 kDa and 300 kDa, or amolecular weight range of between 500 kDa and 2500 kDa.

High molecular weight capsular polysaccharides are able to inducecertain antibody immune responses due to a higher valence of theepitopes present on the antigenic surface. The isolation of “highmolecular weight capsular polysaccharides” is contemplated for use inthe compositions and methods of the present invention. In someembodiments, high molecular weight serotype 5 or 8 capsularpolysaccharide can be isolated and purified ranging from 20 kDa to 1000kDa in molecular weight. In one embodiment, high molecular weightserotype 5 or 8 capsular polysaccharide can be isolated and purifiedranging from 50 kDa to 700 kDa in molecular weight, or ranging from 50kDa to 300 kDa in molecular weight, or ranging from 70 kDa to 300 kDa,or ranging from 90 kDa to 250 kDa, or ranging from 90 kDa to 150 kDa inmolecular weight, or ranging from 90 kDa to 120 kDa in molecular weight,or ranging from 80 kDa to 120 kDa in molecular weight. In someembodiments, a type 5 and/or type 8 capsular polysaccharide oroligosaccharide included in a SA-MAPS immunogenic compositions asdisclosed herein has a high molecular weight of any of 70 kDa to 100 kDain molecular weight; 70 kDa to 110 kDa in molecular weight; 70 kDa to120 kDa in molecular weight; 70 kDa to 130 kDa in molecular weight; 70kDa to 140 kDa in molecular weight; 70 kDa to 150 kDa in molecularweight; 70 kDa to 160 kDa in molecular weight; 80 kDa to 110 kDa inmolecular weight; 80 kDa to 120 kDa in molecular weight; 80 kDa to 130kDa in molecular weight; 80 kDa to 140 kDa in molecular weight; 80 kDato 150 kDa in molecular weight; 80 kDa to 160 kDa in molecular weight;90 kDa to 110 kDa in molecular weight; 90 kDa to 120 kDa in molecularweight; 90 kDa to 130 kDa in molecular weight; 90 kDa to 140 kDa inmolecular weight; 90 kDa to 150 kDa in molecular weight; 90 kDa to 160kDa in molecular weight; 100 kDa to 120 kDa in molecular weight; 100 kDato 130 kDa in molecular weight; 100 kDa to 140 kDa in molecular weight;100 kDa to 150 kDa in molecular weight; 100 kDa to 160 kDa in molecularweight; and similar desired molecular weight ranges. Any whole numberinteger within any of the above ranges is contemplated as an embodimentof the invention.

In one embodiment, the conjugate has a molecular weight of between about50 kDa and about 5000 kDa in molecular weight. In one embodiment, theconjugate has a molecular weight of between about 200 kDa and about 5000kDa in molecular weight. In one embodiment, the immunogenic conjugatehas a molecular weight of between about 500 kDa and about 2500 kDa. Inone embodiment, the immunogenic conjugate has a molecular weight ofbetween about 500 kDa and about 2500 kDa. In one embodiment, theimmunogenic conjugate has a molecular weight of between about 600 kDaand about 2800 kDa. In one embodiment, the immunogenic conjugate has amolecular weight of between about 700 kDa and about 2700 kDa. In oneembodiment, the immunogenic conjugate has a molecular weight of betweenabout 1000 kDa and about 2000 kDa; between about 1800 kDa and about 2500kDa; between about 1100 kDa and about 2200 kDa; between about 1900 kDaand about 2700 kDa; between about 1200 kDa and about 2400 kDa; betweenabout 1700 kDa and about 2600 kDa; between about 1300 kDa and about 2600kDa; between about 1600 kDa and about 3000 kDa. Any whole number integerwithin any of the above ranges is contemplated as an embodiment of theSA-MAPS immunogenic composition as disclosed herein.

In one embodiment, the serotype 5 or 8 capsular polysaccharide has adegree of O-acetylation between 10-100%. In one embodiment, the degreeof O-acetylation is between 50-100%. In one embodiment, the degree ofO-acetylation is between 75-100%. In one embodiment, the immunogenicconjugate generates an antibody that is functional as measured bykilling bacteria in either an animal efficacy model or via anopsonophagocytic killing assay.

Most clinical isolates of S. aureus are encapsulated with eitherserotypes 5 or 8. Type 5 (CP5) and type 8 (CP8) capsular polysaccharides(CPs) have similar tri-saccharide repeating units comprised of N-acetylmannosaminuronic acid, N-acetyl L-fucosamine, and N-acetyl D-fucosamine.See Fournier, J. M. et al., Infect. Immun. 45:97-93 (1984) and Moreau,M., et al, Carbohydrate Res. 201:285-297 (1990). The two CPs, which havethe same sugars, but differ in the sugar linkages and in sites ofO-acetylation, each produce serologically distinct patterns ofimmunoreactivity. CP5 and CP8 are serologically distinct, and this canbe attributed to differences in the linkages between the sugars and inthe sites of O-acetylation.

In some embodiments, a type 5 and/or 8 capsular polysaccharide oroligosaccharide included in a SA-MAPS immunogenic compositions asdisclosed herein can be O-acetylated. In an embodiment, the degree ofO-acetylation of type 5 capsular polysaccharide or oligosaccharide is10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%,90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In an embodiment, the degreeof O-acetylation of type 8 capsular polysaccharide or oligosaccharide is10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%,90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In an embodiment, the degreeof 0-acetylation of type 5 and type 8 capsular polysaccharides oroligosaccharides is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%,60-100%, 70-100%, 80-100%, 90-100%, 50-9%, 60-90%, 70-90% or 80-90%.

The degree of O-acetylation of the polysaccharide or oligosaccharide canbe determined by any method known in the art, for example, by proton NMR(Lemercinier and Jones 1996, Carbohydrate Research 296; 83-96, Jones andLemercinier 2002, J Pharmaceutical and Biomedical analysis 30;1233-1247, WO 05/033148 or WO 00/56357). A further commonly used methodis that described by Hestrin (1949) J. Biol. Chem. 180; 249-261.

O-acetyl groups can be removed by hydrolysis, for example by treatmentwith a base such as anhydrous hydrazine (Konadu et al 1994; Infect.Immun. 62; 5048-5054) or treatment with 0.1N NaOH for 1-8 hours. Inorder to maintain high levels of O-acetylation on type 5 and/or 8polysaccharide or oligosaccharide, treatments which would lead tohydrolysis of the O-acetyl groups are minimized. For example, treatmentat extremes of pH are minimized.

The SA-MAPS immunogenic compositions as disclosed herein comprises, ofconsists essentially of either type 5 or type 8 polysaccharide or aconjugate of type 5 or type 8 polysaccharide. In some embodiments, theSA-MAPS immunogenic compositions as disclosed herein comprise PNAG, ortype 5 or type 8 polysaccharides from S. aureus, where each or all canbe between 30% and 100% 0-acetylated.

In some embodiments, the serotype 5 and/or 8 capsular polysaccharides ofthe SA-MAPS immunogenic composition as disclosed herein are used togenerate antibodies that are functional as measured by the killing ofbacteria in an animal efficacy model or an opsonophagocytic killingassay that demonstrates that the antibodies kill the bacteria. Suchfunctionality may not be observed using an assay that monitors thegeneration of antibodies alone, which is not indicative of theimportance of 0-acetylation in efficacy.

Capsule Epidemiology

The association of particular capsule serotypes with disease is possiblethrough monitoring of clinical isolates. Of the eight differentserotypes of S. aureus identified (Karakawa and Vann (1982) onlyserotypes 1 and 2 are heavily encapsulated, and these are rarelyisolated. See Capsular Polysaccharides of Staphylococcus aureus, p.285-293, In J. B. Robbins, J. C. Hill and J. C. Sadoff (ed.), Seminarsin infectious disease, vol. 4, Bacterial Vaccines. Thieme Stratton, Inc.New York). Surveys have shown that approximately 85-90% of S. aureusclinical isolates express CP5 or CP8 (Arbeit R D, et al., Diagn.Microbiol. Infect. Dis. (1984) April; 2(2):85-91; Karakawa W W, et al.,J. Clin. Microbiol. (1985) September; 22(3):445-7; Essawi T, et al.,Trop. Med. Int. Health. (1998) July; 3(7):576-83; Na'was T, et al., J.Clin. Microbiol. (1998) 36(2):414-20. Most of CP5 and CP8 non-typeablestrains are genetically type 5 or type 8 containing mutations in cap5/8locus (Cocchiaro, Gomez et al., (2006), Mol. Microbiol. February59(3):948-960). Capsulation for some strains is lost rapidly within fewpassages in vitro which is due to a repressive effect of high phosphateconcentration in media used in clinical diagnosis on capsule production.It was also reported that non-capsulated isolates recover capsuleexpression after passing through cows. See Opdebeck, J. P. et al., J.Med. Microbiol. 19:275-278 (1985). Some non-typeable strains becomecapsule positive under appropriate growth conditions.

CP5 and CP8 Structure

The repeat unit of both CP5 and CP8 is comprised of2-acetamido-2-deoxy-D-mannuronic acid, 2-acetamido-2-deoxy-L-fucose and2-acetamido-2-deoxy-D-fucose. See C. Jones et al., Carbohydr. Res.340:1097-1106 (2005). Although CP5 and CP8 have the same sugarcomposition, they have been demonstrated to be immunologically distinct.They differ in glycosidic linkages and site of O-acetylation of uronicacid. Strain dependent incomplete N-acetylation of one of the FucNAcresidues was observed. See Tzianabos et al., PNAS V98: 9365 (2001).

It is important that the S. aureus Capsule Polysaccharide (CP) used inthe SA-MAPS immunogenic composition as disclosed herein is immunogenic.The molecular weight of the S. aureus capsule polysaccharides is animportant consideration, as a high molecular weight capsulepolysaccharide can induce certain antibody immune responses due to ahigher valency of the epitopes present on the antigenic surface. In someembodiments, a CP8 or CP5 used in a SA-MAPS immunogenic composition asdisclosed herein is a high molecular weight capsule polysaccharide type5 and type 8.

Poly N-Acetylated Glucosamine (PNAG)

PNAG is a polysaccharide intercellular adhesion and is composed of apolymer of β-(1→6)-linked glucosamine, optionally substituted withN-acetyl and/or O-succinyl constituents. This polysaccharide is presentin both S. aureus and S. epidermidis and can be isolated from eithersource (Joyce et al 2003, Carbohydrate Research 338; 903; Maira-Litranet al 2002, Infect. Imun. 70; 4433). For example, PNAG may be isolatedfrom S. aureus strain MN8m (WO 04/43407). The preparation of dPNAG isdescribed in WO 04/43405.

The polysaccharide previously known aspoly-N-succinyl-β-(1→6)-glucosamine (PNSG) was recently shown not tohave the expected structure since the identification of N-succinylationwas incorrect (Maira-Litran et al 2002, Infect. Imun. 70; 4433).Therefore, the polysaccharide formally known as PNSG and now found to bePNAG is also encompassed by the term PNAG.

PNAG may be of different sizes varying from over 400 kDa to between 75and 400 kDa to between 10 and 75 kDa to oligosaccharides composed of upto 30 repeat units (of β-(1→6)-linked glucosamine, optionallysubstituted with N-acetyl and O-succinyl constituents). Any size of PNAGpolysaccharide or oligosaccharide may be used in an immunogeniccomposition of the invention, for example a size of over 40 kDa can beused. Sizing may be achieved by any method known in the art, forinstance by microfluidisation, ultrasonic irradiation or by chemicalcleavage (WO 03/53462, EP497524, EP497525).

Size ranges of PNAG are for example 40-400 kDa, 50-350 kDa, 40-300 kDa,60-300 kDa, 50-250 kDa and 60-200 kDa.

PNAG can have different degree of acetylation due to substitution on theamino groups by acetate. PNAG produced in vitro is almost fullysubstituted on amino groups (95-100%). Alternatively, a deacetylatedPNAG can be used having less than 50%, 40%, 30%, 20%, 10% or 5%N-acetylation. Use of a deacetylated PNAG allows opsonic killing of Grampositive bacteria, optionally S. aureus and/or S. epidermidis (WO04/43405). In an embodiment, the PNAG has a size between 40 kDa and 300kDa and is deacetylated so that less than 50%, 40%, 30%, 20%, 10% or 5%of amino groups are N acetylated.

In an embodiment, the PNAG is not 0-succinylated or is O-succinylated onless than 25, 20, 15, 10, 5, 2, 1 or 0.1% of residues.

The term deacetylated PNAG (dPNAG) refers to a PNAG polysaccharide oroligosaccharide in which less than 50%, 40%, 30%, 20%, 10% or 5% of theamino groups are acetylated.

As used herein, the term PNAG encompasses both acetylated anddeacetylated forms of the saccharide.

In an embodiment, PNAG is deacetylated to form dPNAG, by chemicallytreating the native polysaccharide. For example, the native PNAG istreated with a basic solution such that the pH rises to above 10. Forinstance, the PNAG is treated with 0.1-5M, 0.2-4M, 0.3-3M, 0.5-2M,0.75-1.5M or IM NaOH, KOH or NH4OH. Treatment is for at least 10 or 30minutes, or 1, 2, 3, 4, 5, 10, 15 or 20 hours at a temperature of20-100, 25-80, 30-60 or 30-50 or 3545° C. dPNAG may be prepared asdescribed in WO 04/43405.

S. aureus 336 Antigen

In an embodiment, the SA-MAPS immunogenic composition as disclosedherein can comprise the S. aureus 336 antigen described in U.S. Pat. No.6,294,177, which is incorporated herein in its entirety by reference.The 336 antigen comprises β-linked hexosamine, contains no O-acetylgroups and specifically binds to antibodies to S. aureus Type 336deposited under ATCC 55804.

In an embodiment, the 336 antigen is a polysaccharide which is of nativesize or alternatively may be sized, for instance by microfluidisation,ultrasonic irradiation or by chemical treatment. The invention alsocovers oligosaccharides derived from the 336 antigen. The 336 antigen,where included in the immunogenic composition of the invention isoptionally conjugated to a carrier protein as described below or arealternatively unconjugated.

Other Immunogenic Polysaccharides

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein a polysaccharide or oligosaccharidethat is not a S. aureus polysaccharide. For example, in some embodimentsan immunogenic polysaccharide for use in the SA-MAPS complex asdisclosed herein can be a pneumococcal polysaccharide, e.g., a capsularpolysaccharide from Streptococcus pneumoniae from any of the over 93serotypes of pneumococcus that have been identified to date, forexample, including but not limited to serotypes 1, 2, 3, 4, 5, 6A, 6B,6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F, and 33F. Additional pneumococcal serotypes may be identifiedand included in the present SA-MAPS immunogenic composition as describedherein. More than one pneumococcal polysaccharide can be included as thepolymer backbone of the present immunogenic compositions or in a vaccinecomprising the present SA-MAPS composition. In some embodiments, animmunogenic polysaccharide for use in the SA-MAPS complex as disclosedherein is Type 1 capsular polysaccharide (CP1) from Streptococcuspneumoniae.

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein can comprises N. meningitidiscapsular polysaccharides from at least one, two, three or four of theserogroups A, C, W, W135, or Y. In some embodiments, an immunogenicpolysaccharide for use in the SA-MAPS complex as disclosed herein isselected from the group consisting of: Salmonella typhi Vi capsularpolysaccharide, pneumococcal capsular polysaccharides, pneumococcal cellwall polysaccharide, Haemophilus influenzae Type b (Hibb) capsularpolysaccharide, Haemophili polysaccharide, Meningococcal polysaccharide,polysaccharides or oligosaccharides from Gram-positive bacteria (e.g.,Staphylococcus aureus capsular polysaccharide, Bacillus anthracispolysaccharide), Streptococcus polysaccharides (e.g., Gp A and Gp B),Pseudomonas polysaccharide, fungal polysaccharides (e.g., cryptococcyspolysaccharides), viral polysaccharides (e.g., glycoprotein) and otherbacterial capsular or cell wall polysaccharides. In some embodiments, animmunogenic polysaccharide is selected from any of the following,dextran, Vi polysaccharide of Salmonella typhi, pneumococcal capsularpolysaccharide, pneumococcal cell wall polysaccharide (CWPS),meningococcal polysaccharide, Haemophilus influenzae type bpolysaccharide, or any another polysaccharide of viral, prokaryotic, oreukaryotic origin.

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein is selected from the groupconsisting of: Salmonella typhi Vi capsular polysaccharides,pneumococcal capsular polysaccharides, pneumococcal cell wallpolysaccharides, Haemophilus influenzae Type b (Hib) polysaccharides,Haemophili polysaccharides, Meningococcal polysaccharides,polysaccharides or oligosaccharides or lipopolysaccharides fromGram-positive bacteria (e.g., Staphylococcus aureus capsularpolysaccharides, Bacillus anthracis polysaccharides), Streptococcuspolysaccharides (e.g., Gp A and Gp B), Pseudomonas polysaccharides,polysaccharides or oligosaccharides or lipopolysaccharides fromGram-negative bacteria, other bacterial capsular or cell wallpolysaccharides, fungal polysaccharides (e.g., cryptococcuspolysaccharides), viral polysaccharides (e.g., glycoprotein), orpolysaccharides derived from cancer cells.

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein consists of or comprises anantigenic sugar moiety. For example, in some embodiments, apolysaccharide for use in the methods and immunogenic compositions asdisclosed herein is a Vi polysaccharide of Salmonella typhi. The Vicapsular polysaccharide has been developed against bacterial entericinfections, such as typhoid fever. Robbins et al., 150 J. Infect. Dis.436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993).Vi is a polymer of α-1→4-galacturonic acid with an N acetyl at positionC-2 and variable O-acetylation at C-3. The virulence of S. typhicorrelates with the expression of this molecule. Sharma et al., 101 PNAS17492 (2004). The Vi polysaccharide vaccine of S. typhi has severaladvantages: Side effects are infrequent and mild, a single dose yieldsconsistent immunogenicity and efficacy. Vi polysaccharide may bereliably standardized by physicochemical methods verified for otherpolysaccharide vaccines, Vi is stable at room temperature and it may beadministered simultaneously with other vaccines without affectingimmunogenicity and tolerability. Azze et al., 21 Vaccine 2758 (2003).

Thus, the Vi polysaccharide of S. typhi may be cross-linked to a firstaffinity molecule as disclosed herein, for attaching at least oneantigen to the polysaccharide. In some embodiments, the antigen can befrom the same or from another organism, such that the resultingimmunogenic composition confers at least some level of immunity againstone pathogen, or two different pathogens: if the antigen confersprotection against pneumococcus, an immunogenic composition where thepolymer scaffold is a Vi polysaccharide can raise an immunogenicresponse against both S. typhi and pneumococci. Other examples includecombining sugars from encapsulated bacteria (such as meningococcus, S.aureus, pneumococcus, Hib, etc.) and tuberculosis antigens, to providean immunogenic composition that raises an immune response against twodifferent pathogens.

In some embodiments, a polysaccharide for use in the SA-MAPS complex asdisclosed herein is a capsular polysaccharide (CP) or oligosaccharide.In some embodiments, a polysaccharide for use in the SA-MAPS complex asdisclosed herein is a noncapsular polysaccharide or oligosaccharide.

Other immunogenic polysaccharide (PS) for use in the SA-MAPS complex asdisclosed herein can include bacterial cell wall polysaccharides (CWPS),or carbohydrate antigens of cancers.

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein that can serve as a backbone for oneor more SA-antigens or non-SA antigen types are exemplified in Table 2:

TABLE 2 Example immunogenic polysaccharides for the SA-MAPS backbone andassociated example antigens Protein Antigens Polysaccharide Number ofantigens Antigen origins Dextran D90 (60-90KD) Two Staphylococcus aureusD150 (150 KD) Three Staphylococcus aureus D270 (270 KD) ThreeStaphylococcus aureus D500 (425-575 KD) two; three; six Staphylococcusaureus Serotype 1 one, two, three, five pneumococcus, tuberculosis,Pneumococcal Staphylococcus aureus capsular Serotype 3 Fivepneumococcus, tuberculosis, Staphylococcus aureus polysaccharideSerotype 5 one; two; three; five pneumococcus, tuberculosis,Staphylococcus aureus Serotype 6B Two Pneumococcus, Staphylococcusaureus Serotype 7 Three Pneumococcus, Staphylococcus aureus Serotype 14one; two; three; five pneumococcus, tuberculosis Serotype 19 ThreePneumococcus, Staphylococcus aureus Staphylococcus Serotype 5 one; two;three; five, Staphylococcus aureus aureus six Serotype 8 one; two;three; five, Staphylococcus aureus six PNAG one; two; three; five,Staphylococcus aureus six S. aureus 336 one; two; three; five,Staphylococcus aureus antigen six Pneumococcal cell wall polysaccharideFive Pneumococcus, Staphylococcus aureus S. typhi Vi polysaccharide FivePneumococcus, Staphylococcus aureus

In some embodiments, an immunogenic polysaccharide for use in theSA-MAPS complex as disclosed herein can comprise additional polymers,for example, polyethylene glycol-based polymers, poly(ortho ester)polymers, polyacryl carriers, PLGA, polyethylenimine (PEI),polyamidoamine (PAMAM) dendrimers, β-amino ester polymers,polyphosphoester (PPE), liposomes, polymerosomes, nucleic acids,phosphorothioated oligonucleotides, chitosan, silk, polymeric micelles,protein polymers, virus particles, virus-like-particles (VLPs) or othermicro-particles. See, e.g., El-Sayed et al., Smart Polymer Carriers forEnhanced Intracellular Delivery of Therapeutic Molecules, 5 Exp. Op.Biol. Therapy, 23 (2005). Biocompatible polymers developed for nucleicacid delivery may be adapted for use as a backbone herein. See. e.g.,BIOCOMPATIBLE POL. NUCL. ACID. DELIV. (Domb et al., eds., John Wiley &Sons, Inc. Hoboken, N.J., 2011).

For example, VLPs resemble viruses, but are non-infectious because theydo not contain any viral genetic material. The expression, includingrecombinant expression, of viral structural proteins, such as envelopeor capsid components, can result in the self-assembly of VLPs. VLPs havebeen produced from components of a wide variety of virus familiesincluding Parvoviridae (e.g., adeno-associated virus), Retroviridae(e.g., HIV), and Flaviviridae (e.g., Hepatitis B or C viruses). VLPs canbe produced in a variety of cell culture systems including mammaliancell lines, insect cell lines, yeast, and plant cells. Recombinant VLPsare particularly advantageous because the viral component can be fusedto recombinant antigens as described herein.

S. aureus Antigens

It is well recognized that any single animal model of SA infection isunlikely to adequately represent the pathophysiology of disease inhumans; therefore, evaluation of any potential candidate in severalmodels would appear prudent. At the same time, the large number ofvirulence factors (including polysaccharides, surface proteins, andsecreted toxins produced by SA, may provide credence to the idea thatmultiple, genetically conserved antigens should be included in acandidate vaccine. Finally, a closer examination of mechanisms ofimmunity to SA in humans may also provide clues for an effective vaccinestrategy. Indeed, while humoral immunity plays a leading role in hostdefense against many bacterial or viral pathogens, it is unlikely thatantibodies are the only or even the primary factor for resistance to SA.Patients with B-cell deficiencies do not appear to be at significantlyincreased risk of SA infections, and individuals with high levels ofpre-existing SA-specific antibodies can still get infected by SA. On theother hand, a growing body of literature now suggests that T-cellimmunity, the other arm of acquired host defense, plays a critical rolein SA defense. Indeed, individuals with suppressed or impaired cellularimmunity, caused by high dose prednisone therapy, HIV infection,defective interferon-γ (IFN-γ) production, defective interleukin-17(IL-17) production, are at very high risk for SA infection andrecurrence. Moreover, in murine models, IFN-γ or IL-17A/F deficiency hasbeen shown to induce hyper-susceptibility to SA skin infections, andIL-17A deficiency in mice is also associated with prolonged nasalcarriage of SA. Therefore, the inventors have developed a SA-MAPSimmunogenic composition that induces both B- and T-cell acquired immunesresponses in the organism may provide optimal protection against thisorganism.

Herein, the inventors have generated a SA-MAPS immunogenic compositioncomprising containing several conserved SA antigens to elicit a broadrange of immune responses. More specifically, the inventors demonstratea vaccine platform, referred herein as the Staphylococcus aureusMultiple-Antigen-Presenting-System (SA-MAPS), which comprises animmunogenic polysaccharide with affinity-coupled complexes of SAantigens that can induce broad B- and T-cell responses. The immuneresponse generated with the SA-MAPS vaccine was compared to amulti-component SA subunit vaccine using a conventional approach (i.e.,immunization with purified proteins alone, and not attached to animmunogenic polysaccharide). The inventors demonstrated theimmunogenicity of these two vaccines (the antigens alone, or antigens aspart of the SA-MAPS complex) in mice, compared their protective efficacyin SA sepsis infection, dermonecrosis infection, skin abscess infectionand gastrointestinal (GI) colonization models, and finally, studied therole of antigen-specific antibodies and T-cell immunity againstdifferent types of SA infection or colonization

An immunogenic SA antigen for use in the immunogenic compositions andmethods described herein can be any SA antigen, including, but notlimited to pathogenic peptides, toxins, toxoids, subunits thereof, orcombinations thereof. In some embodiments, a SA antigen, which in someembodiments, is fused to the complementary affinity molecule, e.g., abiotin-binding protein such as rhizavidin as disclosed herein, can beany SA. antigen, peptide, polypeptide, polysaccharide, expressed byStaphylococcus aureus bacterium.

In some embodiments, the SA-MAPS comprises at least one or more SAantigens, where the SA antigen is an antigenic protein or polypeptide,and can be selected from any of the group of: hemolysin (Hl) (e.g.,hemolysin α or Hla), Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), Iron regulator surface proteinA (IsdA) and Iron regulator surface protein B (IsdB), or a an antigenicfragment or portion thereof. In some embodiments, the SA-MAPSimmunogenic composition as disclosed herein comprises one or morepeptide or polypeptide fragments of these proteins, as long as theprotein fragment is antigenic, and/or comprises one or more epitopes toinduce an immune response.

Exemplary SA antigens for use in the SA-MAPS composition as disclosedherein can be, for example, but are not limited to: Hla209(27-319), ClfA(221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447).

In some embodiments, a SA-MAPS immunogenic composition as disclosedherein comprises at least 2, or at least 3, or at least 4, or at least5, or all 6 peptide or polypeptide SA-antigens of Hla209(27-319), ClfA(221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447),or proteins or peptides of at least 85% sequence identity thereto. It isenvisioned that any of the above listed SA antigens can be substitutedfor a different SA peptide or polypeptide antigen known to one ofordinary skill in the art. Exemplary SA antigens can be any peptide orpolypeptide comprising at least part of the serine-aspirate repeatprotein E (SdrE) protein, Leukotoxin D (LukD) protein, or Leukotoxin E(LukE) protein, provided that the any peptide or polypeptide isimmunogenic, or is antigenic. Other SA antigens can be used, and aredisclosed herein.

Non-Hemolytic Hemolysin α (Hla)

Hemolysin α (Hla) is a secreted pore-forming toxin and an essentialvirulence factor of MRSA in a mouse model of S. aureus pneumonia. Thelevel of Hla expression by independent S. aureus strains directlycorrelates with their virulence. In some embodiments, the SA antigen isa non-hemolytic Hla, e.g., Hla (209) as disclosed herein.

Hemolysins are exotoxins produced by bacteria that cause lysis of redblood cells. While highly immunogenic, their use in vaccines is limitedbecause they cause lysis of red blood cells. Accordingly, in anotheraspect, provided herein are variants of Staphylococcus aureusalpha-hemolysin (Hla) as the SA antigen for use in the SA-MAPScomposition as disclosed herein, as well as it in a fusion constructwith biotin-binding protein and its uses. These variants, designatedherein as “mHla,” have substantially non-hemolytic, i.e., havesubstantially low hemolytic activity. As used herein, the phrase“substantially non-hemolytic” means an inability to lyse red blood cellsat equivalent titers of wild-type Hla. The term “wild-type Hla” isaccorded the usual definition associated with such phrase, i.e., Hlathat is naturally secreted by a capable bacterial source. “Wild-typeHla,” by definition, does not include, e.g., Hla fusion products derivedvia recombinant DNA techniques. In some embodiments, hemolytic activityof mHla is at least 5%, at least 10%, at least 15%, at least 20%, atleast 20%, at least 30%, at least 30%, at least 35%, least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% lower than an equivalent titers of wild-type Hla. In someembodiments, the mHla has no detectable hemolytic activity. Theinventors have also discovered that hemolytic activity of mHla can befurther reduced by linking the mHla with a biotin-binding protein, e.g.,a rhizavidin biotin-binding protein as disclosed herein. Accordingly,the present disclosure also describes fusion proteins comprising an mHlaprotein and a biotin-binding protein.

In some embodiments, a mHla is where the tripeptide DRD209-211 issubstituted with a tri-alanine peptide (AAA) in the wild-type Hla, andis referred to herein as Hla209 and comprises the following amino acidsequence:

(SEQ ID NO: 16) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN;

In some embodiments, a SA antigen for use in a SA-MAPS immunogeniccomposition as disclosed herein comprises SEQ ID NO: 16, or a protein orpeptide fragment of at least 50 amino acids of SEQ ID NO: 16, or aprotein or peptide having at least 85% amino acid identity to SEQ ID NO:16, where Asp-Arg-Asp (DRD) is mutated to Ala-Ala-Ala (AAA).

In another embodiment, a non-hemolytic Hla can be created where residueW205 or W213 is substituted with alanine (A), and comprise the followingsequences, respectively:

Hla W205A: (SEQ ID NO: 17)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNAGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN;

In some embodiments, a SA antigen for use in a SA-MAPS immunogeniccomposition as disclosed herein comprises SEQ ID NO: 17, or a protein orpeptide fragment of at least 50 amino acids of SEQ ID NO: 17, or aprotein or peptide having at least 85% amino acid identity to SEQ ID NO:17, where amino acid W205 is mutated to Ala (W205A).

HlaW213A: (SEQ ID NO: 18)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSANPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN;

In some embodiments, a SA antigen for use in a SA-MAPS immunogeniccomposition as disclosed herein comprises SEQ ID NO: 18, or a protein orpeptide fragment of at least 50 amino acids of SEQ ID NO: 18, or aprotein or peptide having at least 85% amino acid identity to SEQ ID NO:18, where amino acid W205 is mutated to Ala (W213A).

A non-toxic non-hemolytic mHla protein can be expressed and purified inan E. coli expression system, and the mutants can be made by pointmutation using quick change mutagenesis by a person of ordinary skill inthe art. For example, the nucleotide sequence of a nucleic acid encodingthe wild-type Hla can be changed to replace a given amino acid in thewild-type Hla to another amino acid.

In some embodiments, the Hla variants described herein, e.g., mHla, suchas, Hla209 are ligands for Toll Like Receptors (TLRs), and as such canbe used as TLR ligands. For example, the mHla variants can be used in aSA-MAPS immunogenic composition as disclosed herein can induce TLR2stimulation, e.g., for inducing immunogenicity to otherantigens/pathogens.

In some embodiments, a SA-MAPS immunogenic composition as disclosedherein comprising a mHla SA antigen can elicit an immunologicalresponse-local or systemic. The response can, but need not, beprotective. Accordingly, a non-hemolytic mutant of Hla described hereincan be as an antigen, adjuvant, or a co-stimulator in an immunological,immunogenic, or vaccine composition.

In some embodiments, the antigenic protein is a non-hemolytic Hladescribed herein.

In some embodiments, the non-hemolytic Hla protein is a fusion proteincomprising a biotin-binding protein and a non-hemolytic Hla describedherein.

In alternative embodiments, the Hla antigen is a mutant mHla of H35L(referred to as SEQ ID NO: 5 in US patent application 2011/0274720 whichis incorporated herein in its entirety by reference), which cannot formpores (Menzies, B. E., et al., 1996. Passive immunization with antiserumto a nontoxic alpha-toxin mutant from Staphylococcus aureus isprotective in a murine model. Infect Immun 64:1839-41; Jursch, R., etal., 1994. Histidine residues near the N terminus of staphylococcalalpha-toxin as reporters of regions that are critical foroligomerization and pore formation. Infect Immun 62(6): 2249-56), wasshown to generate antigen-specific immunoglobulin G responses and toafford protection against staphylococcal pneumonia. Transfer ofHla-specific antibodies protects naive animals against S. aureuschallenge and prevents the injury of human lung epithelial cells duringinfection (Bubeck Wardenburg, J., A. M. Palazzolo-Ballance, M. Otto, O,Schneewind, and F. R. DeLeo. 2008. Panton-Valentine leukocidin is not avirulence determinant in murine models of community-associatedmethicillin-resistant Staphylococcus aureus disease. J Infect Dis198:1166-70). To be used as a vaccine, the H35L mutation in Hla isrequired to eliminate toxicity of the protein (Menzies, B. E., and D. S.Kernodle. 1994. Site-directed mutagenesis of the alpha-toxin gene ofStaphylococcus aureus: role of histidines in toxin activity in vitro andin a murine model. Infect Immun 62:1843-7).

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a mHla protein. In certainaspects the mHla protein will have all, or part of the amino acidsequence of SEQ ID NO: 16, e.g., will comprise at least 50, or at least60, or at least 70, or at least 80, or at least 90, or at least 100, orat least 120, or at least 140, or at least 160, or at least 180, or atleast 200, or at least 220 or at least 240, or at least 260, or at least280 amino acids of SEQ ID NO: 16. In one embodiment, a SA antigen of theSA-MAPS immunogenic composition is a polypeptide, peptide, or proteinthat is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical or similar to SEQ ID NO: 16.

The term “Hla protein” refers to a protein that includes isolatedwild-type Hla polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Hla proteins.

Clumping Factor A (ClfA)

Clumping factor A (ClfA) is a S. aureus surface protein associated withbinding to host matrix proteins via a fibrinogen binding site, and isfunctions as a cell wall-associated adhesin protein that mediatesstaphylococcal binding to fibrinogen and platelets. It is expressed onthe cell surface of the bacterium, where it is thought to promotepathogenesis by binding to the fibrinogen and fibrin that is depositedat the site of tissue damage. ClfA is well conserved, and even the mostdiverse form (˜85% identity) exhibits extensive cross-reactivity to bothmonoclonal and polyclonal antibodies.

ClfA is a member of a family of proteins containing the carboxylterminal LPXTG (SEQ ID NO: 19) motif that enables the protein to becomecovalently linked to the cell surface. ClfA also belongs to anotherfamily of proteins (Microbial Surface Components Recognizing AdhesiveMatrix Molecule, or MSCRAMMs) that are associated with binding hostproteins such as fibrinogen (bound by ClfA), the fibronectin bindingproteins (FnbA and FnbB), the collagen binding protein (Cna) and others.These proteins all share the amino terminal signal sequence thatmediates transport to the cell surface. The MSCRAMMs also include anA-domain that is the functional region containing the active site forligand binding (e.g., fibrinogen, fibronectin, elastin, keratin). TheA-domain is followed by a region composed of serine aspartate repeats(SD repeat), which is thought to span the peptidoglycan layer. The SDrepeat is followed by a membrane-spanning region that includes the LPXTG(SEQ ID NO: 19) motif for covalent linkage of the protein topeptidoglycan. ClfA is described in U.S. Pat. No. 6,008,341.

Thus, ClfA is a reasonable candidate for a component of a vaccineagainst S. aureus. However, given the structural instability of ClfA, aformulation of ClfA is problematic since it can readily degrade overtime in storage.

Full-length ClfA comprises several regions and domains: an N-terminalsecretory domain (“S” domain); followed by a ligand-binding A region,which contains three domains (N1, N2, which contains an EF-hand motif,and N3); followed by an R region, which contains serine-aspartatedipeptide repeats; followed by a cell wall-binding region (“W” region)containing an LPXTG motif; a hydrophobic membrane-spanning domain (“M”region); and a charged C-terminus (“C” region) containing positivelycharged amino acids. The Nl region contains a protease-sensitive site.Much of the instability of ClfA is attributed to the clipping of ClfA atNl, which results in fragments containing Nl and N2N3.

The structure and function of ClfA is disclosed in U.S. PatentApplication Publication No. 2007/0087014A 1 (Pavliak et al, Apr. 19,2007), and U.S. Pat. No. 6,008,341 which are incorporated herein byreference in their entirety.

ClfA contains a protease resistant domain which is used forimmunization. Passive immunization of mice with anti-ClfA and anti CP5antibodies effectively sterilized mammary glands in mammary glandinfection model (Tuchscherr, L. P., F. R. Buzzola, L. P. Alvarez, J. C.Lee, and D. O. Sordelli. 2008. Antibodies to capsular polysaccharide andclumping factor A prevent mastitis and the emergence of unencapsulatedand small-colony variants of Staphylococcus aureus in mice. Infect Immun76: 5738-44).

The ligand binding region of ClfA comprising N1N2N3 of the A domainspans amino acids 40-559. The N domains of ClfA have been assigned asfollows: N1 encompasses residues 45-220; N2 encompasses residues229-369; and N3 encompasses residues 370-559. See Deivanayagam et al.EMBO J. 21:6660-6672 (2002). For ease of reference the N1N2N3 domainsmay be referred to as N123, likewise N2N3 may be referred to as N23. Inpreparations of recombinant N1N2N3, the N1 domain has been found to beprotease sensitive and is easily cleaved or hydrolyzed to leave the N2N3as a stable ligand binding recombinant fragment. See Deivanayagam et al.EMBO J. 21:6660-6672 (2002). The crystal structure of the fibrinogenbinding N2N3 fragment of ClfA A domain, revealed that both N2 and N3 aredominated by anti-parallel beta strands. In addition to theanti-parallel beta strands, the N2 domain contains a single turn alphahelix and two 3₁₀ helices and the N3 domain contains three 3₁₀ helices.See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). Sequence alignmentof N2 and N3 reveals only 13% sequence identity and 36% sequencesimilarity over their lengths. See Deivanayagam et al. EMBO J.21:6660-6672 (2002). The topology of the N2 and N3 domains are similarto the classic IgG fold and have been proposed to be novel variants ofthe IgG fold. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002).

ClfA Sequence: The gene for clumping factor protein A, designated ClfA,has been cloned, sequenced and analyzed in detail at the molecular level(McDevitt et al., Mol. Microbiol. 11: 237-248 (1994); McDevitt et al.,Mol. Microbiol. 16:895-907 (1995)).

In some embodiments, the ClfA antigen for use in the SA-MAPS immunogeniccomposition as disclosed herein comprises a polypeptide or peptidecomprising at least part of SEQ ID NO: 2, which corresponds to the fulllength ClfA mature protein from S. aureus strain USA300 (without thesignal sequence).

(SEQ ID NO: 2) SENSVTQSDSASNESKSNDSSSVSAAPKTDDTNVSDTKTSSNTNNGETSVAQNPAQQETTQSSSTNATTEETPVTGEATTTTTNQANTPATTQSSNTNAEELVNQTSNETTFNDTNTVSSVNSPQNSTNAENVSTTQDTSTEATPSNNESAPQSTDASNKDVVNQAVNTSAPRMRAFSLAAVAADAPAAGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFEDVTNSVNITFPNPNQYKVEENTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDKPVVPEQPDEPGEIEPIPEDSDSDPGSDSGSDSNSDSGSDSGSDSTSDSGSDSASDSDSASDSDSASDSDSASDSDSASDSDSDNDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSASDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSESDSDSESDSDSDSDSDSDSDSDSDSDSDSASDSDSGSDSDSSSDSDSESDSNSDSESGSNNNVVPPNSPKNGTNASNKNEAKDSKEPLPDTGSEDEANTSLIWGLLASIGSLL LFRRKKENKDKK

In some embodiments, the ClfA antigen for use in the SA-MAPS immunogeniccomposition as disclosed herein is ClfA (221-559) (SEQ ID NO: 3), or afragment or protein of at least 85% amino acid sequence identitythereto. SEQ ID NO: 3 has the following amino acid sequence:

(SEQ ID NO: 3) VAADAPAAGT DITNQLTNVT VGIDSGTTVY PHQAGYVKLNYGFSVPNSAV KGDTFKITVP KELNLNGVTS TAKVPPIMAGDQVLANGVID SDGNVIYTFT DYVNTKDDVK ATLTMPAYIDPENVKKTGNV TLATGIGSTT ANKTVLVDYE KYGKFYNLSIKGTIDQIDKT NNTYRQTIYV NPSGDNVIAP VLTGNLKPNTDSNALIDQQN TSIKVYKVDN AADLSESYFV NPENFEDVTNSVNITFPNPN QYKVEFNTPD DQITTPYIVV VNGHIDPNSKGDLALRSTLY GYNSNIIWRS MSWDNEVAFN NGSGSGDGID KPVVPEQPDE PGEIEPIPE

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a ClfA of SEQ ID NO: 2 orSEQ ID NO: 3. In certain aspects a ClfA antigen peptide or polypeptidewill have all, or part of the amino acid sequence of SEQ ID NO: 3, e.g.,will comprise at least 50, or at least 60, or at least 70, or at least80, or at least 90, or at least 100, or at least 120, or at least 140,or at least 160, or at least 180, or at least 200, or at least 220 or atleast 240 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. In oneembodiment, a ClfA antigen peptide or polypeptide present in the SA-MAPSimmunogenic composition is a polypeptide, peptide, or protein that is oris at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical or similar to SEQ ID NO: 2.

In alternative embodiments, a ClfA antigen for use in the SA-MAPSimmunogenic composition as disclosed herein is a protein or peptidehaving an amino acid sequence of ClfA from 111 S. aureus disease-causingisolates disclosed in Table 10 of U.S. Pat. No. 8,568,735, which isincorporated herein in its entirety by reference. In some embodiments, aClfA antigen for use in the SA-MAPS immunogenic composition as disclosedherein is a ClfA variant of SEQ ID NO: 61-108, or a mutant ClfA from S.aureus strain PFESA0237 of SEQ ID NO: 130, 131 and 123 as disclosed inU.S. Pat. No. 8,568,735, and are encompassed for use in the SA-MAPSimmunogenic composition as disclosed herein.

The amino acid sequence of the full length (including the signalsequence) wild type ClfA from S. aureus strain PFESA0237 is disclosed asSEQ ID NO: 130 in U.S. Pat. No. 8,568,735. SEQ ID NO: 130 has a tyrosineat position 338, which is changed to an alanine in the mutated form ofClfA (mClfA). The full length gene encoding the wild type ClfA from S.aureus strain PFESA0237, comprising the N123 region, the repeat regionand the anchor region is disclosed as SEQ ID NO: 131 in U.S. Pat. No.8,568,735, and the amino acid sequence of the mClfA(Y338A) id disclosedas SEQ ID NO: 123 in U.S. Pat. No. 8,568,735. However, it should benoted that the change from a tyrosine to an alanine, which occurs in thewild type ClfA at position 338 of SEQ ID NO: 130, and which isdesignated as Y338A, is shown in the mutated form of ClfA, in SEQ ID NO:123 at position 310. Furthermore, the mutated form of ClfA shown in theamino acid sequence of SEQ ID NO: 123 is the mature form of ClfA withoutthe signal sequence, thus accounting for the difference in position ofthis mutation between SEQ ID NO: 130 and SEQ ID NO: 123.

The term “ClfA protein” refers to a protein that includes isolatedwild-type ClfA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria ClfA proteins.

Clumping Factor B (ClfB)

Clumping factor B (ClfB) is a S. aureus protein having fibrinogenbinding activity and triggers S. aureus to form clumps in the presenceof plasma. ClfB is an MSCRAMM protein and displays the characteristicMSCRAMM domain organization including an A-domain that is the functionalregion containing the active site for ligand binding (e.g., fibrinogen,fibronectin, elastin, keratin). The A-domain is followed by a regioncomposed of serine aspartate repeats (SD repeat), which is thought tospan the peptidoglycan layer. The SD repeat is followed by amembrane-spanning region that includes the LPXTG (SEQ ID NO: 19) motiffor covalent linkage of the protein to peptidoglycan. ClfB is describedin WO 99/27109 and in U.S. Pat. Nos. 6,680,195 and 8,568,735, which areincorporated herein in their entirety by reference.

The internal organization of ClfB N-terminal A domain is very similarorganization as found in ClfB. The A domain is composed of threesubdomains N1, N2, and N3. The ligand binding region of ClfB comprisingN1N2N3 of the A domain (FIG. 1) spans amino acids 44-585. For ease ofreference the N1N2N3 domains may be referred to as N123, likewise N2N3may be referred to as N23. The N domains of ClfB have been assigned asfollows: N1 encompasses residues 44-197; N2 encompasses residues198-375; and N3 encompasses residues 375-585. In ClfA, the crystalstructure of the A domain was found to have a unique version of theimmunoglobulin fold and by analogy the same may be speculated to be thecase for ClfB. See Deivanayagam et al., EMBO J. 21:6660-6672 (2002).Even though organization of the A domains of ClfB and ClfA are similar,sequence identity is only 260, See Ni Eidhin et al., Mol. Microbiol.30:245-257 (2002).

ClfB Sequence: The gene encoding ClfB is classified as a core adhesiongene. In some embodiments, the ClfB antigen for use in the SA-MAPSimmunogenic composition as disclosed herein comprises a polypeptide orpeptide comprising at least part of SEQ ID NO: 4, which corresponds tothe full length ClfB mature protein from S. aureus strain USA300(without the signal sequence).

(SEQ ID NO: 4) SEQSNDTTQSSKNNASADSEKNNMIETPQLNTTANDTSDISANTNSANVDSTTKPMSTQTSNTTTTEPASTNETPQPTAIKNQATAAKMQDQTVPQEANSQVDNKTTNDANSIATNSELKNSQTLDLPQSSPQTISNAQGTSKPSVRTRAVRSLAVAEPVVNAADAKGTNVNDKVTASNFKLEKTTFDPNQSGNTFMAANFTVTDKVKSGDYFTAKLPDSLTGNGDVDYSNSNNTMPIADIKSTNGDVVAKATYDILTKTYTFVFTDYVNNKENINGQFSLPLFTDRAKAPKSGTYDANINIADEMFNNKITYNYSSPIAGIDKPNGANISSQIIGVDTASGQNTYKQTVFVNPKQRVLGNTWVYIKGYQDKIEESSGKVSATDTKLRIFEVNDISKLSDSYYADPNDSNLKEVTDQFKNRIYYEHPNVASIKFGDITKTYVVLVEGHYDNTGKNLKTQVIQENVDPVTNRDYSIFGWNNENVVRYGGGSADGDSAVNPKDPTPGPPVDPEPSPDPEPEPSPDPDPDSDSDSDSGSDSDSGSDSDSESDSDSDSDSDSDSDSDSESDSDSESDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSESDSDSESDSESDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSRVTPPNNEQKAPSNPKGEVNHSNKVSKQHKTDALPETGDKSENTNATLFGA MMALLGSLLLFRKRKQDHKEKA

In some embodiments, the ClfB antigen for use in the SA-MAPS immunogeniccomposition as disclosed herein is ClfB (203-542) (SEQ ID NO: 5), or afragment or protein of at least 85% amino acid sequence identitythereto. SEQ ID NO: 5 has the following amino acid sequence:

(SEQ ID NO: 5) PVVNAADAKGTNVNDKVTASNFKLEKTTFDPNQSGNTFMAANETVTDKVKSGDYETAKLPDSLTGNGDVDYSNSNNTMPIADIKSTNGDVVAKATYDILTKTYTFVFTDYVNNKENINGQFSLPLFTDRAKAPKSGTYDANINIADEMFNNKITYNYSSPIAGIDKPNGANISSQIIGVDTASGQNTYKQTVFVNPKQRVLGNTWVYIKGYQDKIEESSGKVSATDTKLRIFEVNDISKLSDSYYADPNDSNLKEVTDQFKNRIYYEHPNVASIKFGDITKTYVVLVEGHYDNTGKNLKTQVIQENVDPVTNRDYSIFGWNNENVVRYGGGSADGDSAVN

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a ClfB of SEQ ID NO: 4 orSEQ ID NO: 5. In certain aspects a ClfB antigen peptide or polypeptidewill have all, or part of the amino acid sequence of SEQ ID NO: 5, e.g.,will comprise at least 50, or at least 60, or at least 70, or at least80, or at least 90, or at least 100, or at least 120, or at least 140,or at least 160, or at least 180, or at least 200, or at least 220 or atleast 240 amino acids of SEQ ID NO: 4 or SEQ ID NO: 5. In oneembodiment, a ClfB antigen peptide or polypeptide present in the SA-MAPSimmunogenic composition is a polypeptide, peptide, or protein that is oris at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical or similar to SEQ ID NO: 5.

In alternative embodiments, a ClfB antigen for use in the SA-MAPSimmunogenic composition as disclosed herein is a protein or peptidehaving an amino acid sequence of one the ClfB proteins sequenced from 92strains of S. aureus associated with multiple disease states, which aredisclosed in Table 11 of U.S. Pat. No. 8,568,735. Other ClfB antigensnot identified herein are disclosed encompassed for use in the SA-MAPSimmunogenic composition, provided they are antigenic.

The term “ClfB protein” refers to a protein that includes isolatedwild-type ClfB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria ClfB proteins.

Serine-Aspirate Repeat Protein D (SdrD)

SdrD Sequence: In some embodiments, the SdrD antigen for use in theSA-MAPS immunogenic composition as disclosed herein comprises apolypeptide or peptide comprising at least part of SEQ ID NO: 6, whichcorresponds to the full length SdrD mature protein (aa53-1831) from S.aureus strain USA300 (without the signal sequence).

(SEQ ID NO: 6) AESTNKELNEATTSASDNQSSDKVDMQQLNQEDNTKNDNQKEMVSSQGNETTSNGNKLIEKESVQSTTGNKVEVSTAKSDEQASPKSTNEDLNTKQTISNQEALQPDLQENKSVVNVQPTNEENKKVDAKTESTTLNVKSDAIKSNDETLVDNNSNSNNENNADIILPKSTAPKRLNTRMRIAAVQPSSTEAKNVNDLITSNTTLTVVDADKNNKIVPAQDYLSLKSQITVDDKVKSGDYFTIKYSDTVQVYGLNPEDIKNIGDIKDPNNGETIATAKHDTANNLITYTFTDYVDRFNSVQMGINYSIYMDADTIPVSKNDVEFNVTIGNTTTKTTANIQYPDYVVNEKNSIGSAFTETVSHVGNKENPGYYKQTIYVNPSENSLTNAKLKVAAYHSSYPNNIGQINKDVTDIKIYQVPKGYTLNKGYDVNTKELTDVTNQYLQKITYGDNNSAVIDFGNADSAYVVMVNTKFQYTNSESPTLVQMATLSSTGNKSVSTGNALGFTNNQSGGAGQEVYKIGNYVWEDTNKNGVQELGEKGVGNVTVTVFDNNTNTKVGEAVTKEDGSYLIPNLPNGDYRVEFSNLPKGYEVTPSKQGNNEELDSNGLSSVITVNGKDNLSADLGIYKPKYNLGDYVWEDTNKNGIQDQDEKGISGVTVTLKDENGNVLKTVTTDADGKYKFTDLDNGNYKVEFTTPEGYTPTTVTSGSDIEKDSNGLTTTGVINGADNMTLDSGFYKTPKYNLGNYVWEDTNKDGKQDSTEKGISGVTVTLKNENGEVLQTTKTDKDGKYQFTGLENGTYKVEFETPSGYTPTQVGSGTDEGIDSNGTSTTGVIKDKDNDTIDSGFYKPTYNLGDYVWEDTNKNGVQDKDEKGISGVTVTLKDENDKVLKTVTTDENGKYQFTDLNNGTYKVEFETPSGYTPTSVTSGNDTEKDSNGLTTTGVIKDADNMTLDSGFYKTPKYSLGDYVWYDSNKDGKQDSTEKGIKDVKVTLLNEKGEVIGTTKTDENGKYCFDNLDSGKYKVIFEKPAGLTQTGTNTTEDDKDADGGEVDVTITDHDDFTLDNGYYEEETSDSDSDSDSDSDSDRDSDSDSDSDSDSDSDSDSDSDSDSDSDSDRDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDAGKHTPVKPMSTTKDHHNKAKALPETGNENSGSNNATLFGGLFAALGSLLLFGR RKKQNK 

In some embodiments, the SdrD antigen for use in the SA-MAPS immunogeniccomposition as disclosed herein is SdrD (246-682) (SEQ ID NO: 7), or afragment or protein of at least 85% amino acid sequence identitythereto. SEQ ID NO: 7 has the following amino acid sequence:

(SEQ ID NO: 7) NVNDLITSNTTLTVVDADKNNKIVPAQDYLSLKSQITVDDKVKSGDYFTIKYSDTVQVYGLNPEDIKNIGDIKDPNNGETIATAKHDTANNLITYTFTDYVDRFNSVQMGINYSIYMDADTIPVSKNDVEFNVTIGNTTTKTTANIQYPDYVVNEKNSIGSAFTETVSHVGNKENPGYYKQTIYVNPSENSLTNAKLKVQAYHSSYPNNIGQINKDVTDIKIYQVPKGYTLNKGYDVNTKELTDVTNQYLQKITYGDNNSAVIDFGNADSAYVVMVNTKFQYTNSESPTLVQMATLSSTGNKSVSTGNALGFTNNQSGGAGQEVYKIGNYVWEDTNKNGVQELGEKGVGNVTVTVFDNNTNTKVGEAVTKEDGSYLIPNLPNGDYRVEFSNLPKGYEVTPSKQGNNEELDSNGLSSVITVNGKDNLSADLGIYKPKY

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a SdrD of SEQ ID NO: 6 orSEQ ID NO: 7. In certain aspects a SdrD antigen peptide or polypeptidewill have all, or part of the amino acid sequence of SEQ ID NO: 7, e.g.,will comprise at least 50, or at least 60, or at least 70, or at least80, or at least 90, or at least 100, or at least 120, or at least 140,or at least 160, or at least 180, or at least 200, or at least 220 or atleast 240 amino acids of SEQ ID NO: 6 or SEQ ID NO: 7. In oneembodiment, a SdrD antigen peptide or polypeptide present in the SA-MAPSimmunogenic composition is a polypeptide, peptide, or protein that is oris at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical or similar to SEQ ID NO: 7.

The term “SdrD protein” refers to a protein that includes isolatedwild-type SdrD polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SdrD proteins.

In some embodiments, other SdrD antigens can be used in the SA-MAPScomposition as disclosed herein, e.g., SdrD antigenic proteins orpeptides derived from various species of organisms, some of whichinclude the following SdrD from S. aureus: strain USA300 FPR3757(protein accession number SAUSA300 0547); strain NCTC8325 (proteinaccession number SAOUHSC 00545): strain MW2 (protein accession numberMW0517); strain MSSA476 (protein accession number SAS0520; and strainMu50 (protein accession number SAV0562).

Iron Regulator Surface Protein A (IsdA)

IsdA Sequence: In some embodiments, the IsdA antigen for use in theSA-MAPS immunogenic composition as disclosed herein comprises apolypeptide or peptide comprising at least part of SEQ ID NO: 10, whichcorresponds to the full length IsdA mature protein (aa 47-350) from S.aureus strain USA300 (without the signal sequence).

(SEQ ID NO: 10) ATEATNATNNQSTQVSQATSQPINFQVQKDGSSEKSHMDDYMQHPGKVIKQNNKYYFQTVLNNASFWKEYKFYNANNQELATTVVNDNKKADTRTINVAVEPGYKSLTTKVHIVVPQINYNHRYTTHLEFEKAIPTLADAAKPNNVKPVQPKPAQPKTPTEQTKPVQPKVEKVKPTVTTTSKVEDNHSTKVVSTDTTKDQTKTQTAHTVKTAQTAQEQNKVQTPVKDVATAKSESNNQAVSDNKSQQTNKVTKHNETPKQASKAKELPKTGLTSVDNFISTVAFATLALLGSLS LLLFKRKESK

In some embodiments, the IsdA antigen for use in the SA-MAPS immunogeniccomposition as disclosed herein is IsdA (47-324) (SEQ ID NO: 11), or afragment or protein of at least 85% amino acid sequence identitythereto. SEQ ID NO: 11 has the following amino acid sequence:

(SEQ ID NO: 11) ATEATNATNNQSTQVSQATSQPINFQVQKDGSSEKSHMDDYMQHPGKVIKQNNKYYFQTVLNNASFWKEYKFYNANNQELATTVVNDNKKADTRTINVAVEPGYKSLTTKVHIVVPQINYNHRYTTHLEFEKAIPTLADAAKPNNVKPVQPKPAQPKTPTEQTKPVQPKVEKVKPTVTTTSKVEDNHSTKVVSTDTTKDQTKTQTAHTVKTAQTAQEQNKVQTPVKDVATAKSESNNQAVSDNKSQQTNKVTKHNETPKQASKAKELPKTGLTSVDNF 

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a IsdA of SEQ ID NO: 10 orSEQ ID NO: 11. In certain aspects a IsdA antigen peptide or polypeptidewill have all, or part of the amino acid sequence of SEQ ID NO: 11,e.g., will comprise at least 50, or at least 60, or at least 70, or atleast 80, or at least 90, or at least 100, or at least 120, or at least140, or at least 160, or at least 180, or at least 200, or at least 220or at least 240 amino acids of SEQ ID NO: 10 or SEQ ID NO: 11. In oneembodiment, a IsdA antigen peptide or polypeptide present in the SA-MAPSimmunogenic composition is a polypeptide, peptide, or protein that is oris at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical or similar to SEQ ID NO: 11.

The term “IsdA protein” refers to a protein that includes isolatedwild-type IsdA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria IsdA proteins.

Iron Regulator Surface Protein B (IsdB)

In some embodiments, a SA antigen for use in the SA-MAPS composition asdisclosed herein is the S. aureus surface protein iron surfacedeterminant B (IsdB). his MSCRAMM was described by Mazmanian et al.(Mazmanian, S K et al. Proc. Natl. Acad. Sci., USA 99:2293-2298 (2002))and it has subsequently been tested and shown to be effective as avaccine candidate in a murine model of infection and a rhesus macaqueimmunogenicity study by Kuklin, et al. (Kuklin, N A, et al. Infectionand Immunity, Vol. 74, No. 4, 2215-2223, (2006)).

IsdB Sequence: In some embodiments, the IsdB antigen for use in theSA-MAPS immunogenic composition as disclosed herein comprises apolypeptide or peptide comprising at least part of SEQ ID NO: 12, whichcorresponds to the full length IsdB mature protein (aa 41-652) from S.aureus strain USA300 (without the signal sequence).

(SEQ ID NO: 12) AAEETGGTNTEAQPKTEAVASPTTTSEKAPETKPVANAVSVSNKEVEAPTSETKEAKEVKEVKAPKETKEVKPAAKATNNTYPILNQELREAIKNPAIKDKDHSAPNSRPIDFEMKKKDGTQQFYHYASSVKPARVIFTDSKPEIELGLQSGQFWRKFEVYEGDKKLPIKLVSYDTVKDYAYIRFSVSNGTKAVKIVSSTHFNNKEEKYDYTLMEFAQPIYNSADKFKTEEDYKAEKLLAPYKKAKTLERQVYELNKIQDKLPEKLKAEYKKKLEDTKKALDEQVKSAITEFQNVQPTNEKMTDLQDTKYVVYESVENNESMMDTFVKHPIKTGMLNGKKYMVMETTNDDYWKDFMVEGQRVRTISKDAKNNTRTIIFPYVEGKTLYDAIVKVHVKTIDYDGQYHVRIVDKEAFTKANTDKSNKKEQQDNSAKKEATPATPSKPTPSPVEKESQKQDSQKDDNKQLPSVEKENDASSESGKDKTPATKPTKGEVESSSTTPTKVVSTTQNVAKPTTASSKTTKDVVQTSAGSSEAKDSAPLQKANIKNTNDGHTQSQNNKNTQENKAKSLPQTGEESNKDMTLPLMAL LALSSIVAFVLPRKRKN

In some embodiments, the IsdB antigen for use in the SA-MAPS immunogeniccomposition as disclosed herein is IsdB (48-477) (SEQ ID NO: 13), or afragment or protein of at least 85% amino acid sequence identitythereto. SEQ ID NO: 13 has the following amino acid sequence:

(SEQ ID NO: 13) TNTEAQPKTEAVASPTTTSEKAPETKPVANAVSVSNKEVEAPTSETKEAKEVKEVKAPKETKEVKPAAKATNNTYPILNQELREAIKNPAIKDKDHSAPNSRPIDFEMKKKDGTQQFYHYASSVKPARVIFTDSKPEIELGLQSGQFWRKFEVYEGDKKLPIKLVSYDTVKDYAYIRFSVSNGTKAVKIVSSTHFNNKEEKYDYTLMEFAQPIYNSADKFKTEEDYKAEKLLAPYKKAKTLERQVYELNKIQDKLPEKLKAEYKKKLEDTKKALDEQVKSAITEFQNVQPTNEKMTDLQDTKYVVYESVENNESMMDTFVKHPIKTGMLNGKKYMVMETTNDDYWKDFMVEGQRVRTISKDAKNNTRTIIFPYVEGKTLYDAIVKVHVKTIDYDGQYHVRIVDKEAFTKANTDKSNKKEQQDNSAKKEAT

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a IsdB of SEQ ID NO: 12 orSEQ ID NO: 13. In certain aspects a IsdA antigen peptide or polypeptidewill have all, or part of the amino acid sequence of SEQ ID NO: 13,e.g., will comprise at least 50, or at least 60, or at least 70, or atleast 80, or at least 90, or at least 100, or at least 120, or at least140, or at least 160, or at least 180, or at least 200, or at least 220or at least 240 amino acids of SEQ ID NO: 12 or SEQ ID NO: 31. In oneembodiment, a IsdB antigen peptide or polypeptide present in the SA-MAPSimmunogenic composition is a polypeptide, peptide, or protein that is oris at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical or similar to SEQ ID NO: 13.

The term “IsdB protein” refers to a protein that includes isolatedwild-type IsdB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria IsdB proteins.

In some embodiments, other IsdB antigens can be used in the SA-MAPScomposition as disclosed herein, e.g., IsdB antigenic proteins orpeptides derived from various species of organisms, some of whichinclude the following IsdB from S. aureus strains, including strainMRSA252 (protein accession number CAG40104.1); strain Newman (proteinaccession number BAF67312.1); strain MSSA476 (protein accession numberCAG42837.1); strain Mu3 (protein accession number BAF78003.1); strainRF122 (protein accession number CAI80681.1).

Other SA Antigens

While exemplary SA antigens used in the SA-MAPS composition as disclosedherein can be one or more of, or all 6 of hemolysin (Hl) (e.g.,hemolysin α or Hla209), Clumping factor A (ClfA), Clumping factor B(ClfB), serine-aspirate repeat protein D (SdrD), Iron regulator surfaceprotein A (IsdA) and Iron regulator surface protein B (IsdB), orfragments thereof, e.g., Hla209(27-319), ClfA (221-559), ClfB (203-542),SdrD (246-682), IsdA (47-324), IsdB (48-447) or proteins or peptideshaving at least 85% sequence identity thereto, it is envisioned that anyof the above listed SA antigens can be substituted for a different SApeptide or polypeptide antigen known to one of ordinary skill in theart.

For example, in some embodiments, any one or more SA antigens useful inthe SA-MAPS composition as disclosed herein include, but are not limitedto, a peptide or polypeptide comprising at least part of theserine-aspirate repeat protein E (SdrE) protein, SdrC, Leukotoxin D(LukD) protein, or Leukotoxin E (LukE) protein, provided that the anypeptide or polypeptide is immunogenic, or is antigenic. Other SAantigens can be used, and are disclosed herein.

In some embodiments, other SA antigens can be used in the SA-MAPScomposition as disclosed herein. For example, the S. aureus MntC protein(also known as Protein 305, P305, P305A, and ORF305) is a component of amanganese ABC transporter. This protein is expressed in vivo. S. aureususes manganese as a cofactor for an enzyme that enhances the survival ofS. aureus in neutraphils. MntC is, therefore, important for the in vivosurvival of S. aureus during infection. Like ClfA, this protein is alsounstable in solution. However, unlike ClfA, which can aggregate, or clipvia hydrolysis, the primary mechanism of MntC degradation is deamidationwhen subject to basic pH and/or temperature around room temperature(about 25° C.) or higher.

In some embodiments SA antigens can be used in the SA-MAPS compositionas disclosed herein can be selected from any one or, or a combinationof: SdrC, SdrE, MntC/SitC/Saliva Binding Protein, Opp3a, DltA, HtsA,LtaS, SdrH, SrtA, SpA, SBI, beta-hemolysin, fibronectin-binding proteinA (fnbA), coagulase, map, Panton-Valentine leukocidin (pvl), gamma-toxin(hlg), ica, immunodominant ABC transporter, RAP, autolysin, lamininreceptors, SPOIIIE, SsaA, EbpS, Sasf, SasH, EFB (FIB), FnbB, Npase, EBP,bone sialo binding protein II; aureolysin precursor (AUR)/Sepp1, Cna,TSST-1, mecA, dPNAG, GehD, EbhA, EbhB, SSP-1, SSP-2 HBP, vitronectinbinding protein, HarA, Enterotoxin A, Enterotoxin B, Enterotoxin C1, andnovel autolysin.

In some embodiments SA antigens can be used in the SA-MAPS compositionas disclosed herein can be selected from any one or, or a combination ofOpp3a, DltD, HtsA, LtaS, IsdA, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Shialpha-hemolysin (hla), beta-hemolysin, fibronectin-binding protein A(fnbA), fibronectin-binding protein B (fnbB), coagulase, Fig, map,Panton-Valentine leukocidin (pvl), alpha-toxin and its variants,gamma-toxin (hlg) and variants, ica, immunodominant ABC transporter,Mg2+ transporter, Ni ABC transporter, RAP, autolysin, laminin receptors,IsaA/PisA, IsaB/PisB, SPOIIIE, SsaA, EbpS, SasA, SasF, SasH, EFB (FIB),SBI, Npase, EBP, bone sialo binding protein II, aureolysin precursor(AUR)/Sepp1, Cna, and fragments thereof such as M55, TSST-1, mecA,poly-N-acetylglucosamine (PNAG/dPNAG) exopolysaccharide, GehD, EbhA,EbhB, SSP-1, SSP-2, HBP, vitronectin binding protein, HarA, EsxA, EsxB,Enterotoxin A, Enterotoxin B. Enterotoxin C1, and novel autolysin.

Bacterial antigens include, but are not limited to (i) a secretedvirulence factor, and/or a cell surface protein or peptide, or (ii) arecombinant nucleic acid molecule encoding a secreted virulence factor,and/or a cell surface protein or peptide. The bacterial antigen caninclude one or more of at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 additional staphylococcalantigen or immunogenic fragment thereof, including, but not limited toFnBpA, FnBpB, LukD (GI:2765304), LukE (GI:2765303), LukF (GI:12231006),SasA, SasD, SasG, SasI, SasK, SpA (and variants thereof), Eap, Ebh, Emp,EsaB, EsaC, EsxA, EsxB, SdrC, SdrE, Coa, Hla (e.g., H35 mutants), IsdC,SasF, vWbp, vWh, 52 kDa vitronectin binding protein (WO 01/60852), Aaa(GenBank CAC80837), Aap (GenBank accession AJ249487), Ant (GenBankaccession NP-372518), autolysin glucosaminidase, autolysin amidase, Cna,collagen binding protein (U.S. Pat. No. 6,288,214), EFB (FIB), Elastinbinding protein (EbpS), EPB, FbpA, fibrinogen binding protein (U.S. Pat.No. 6,008,341), Fibronectin binding protein (U.S. Pat. No. 5,840,846),FnbA, FnbB, GehD (US 2002/0169288), HarA, HBP, Immunodominant ABCtransporter, IsaA/P isA, laminin receptor, Lipase GehD, MAP, Mg2+transporter, MHC II analogue (U.S. Pat. No. 5,648,240), MRPII, Npase,RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI,SdrF (WO 00/12689), SdrG/Fig (WO 00/12689), SdrH (WO 00/12689), SEAexotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABCtransporter, SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234),SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein (see PCTpublications WO2007/113222, WO2007/113223, WO2006/032472, WO2006/032475,WO2006/032500, each of which is incorporated herein by reference intheir entirety).

In some embodiments, a SA-antigen for use in the SA-MAPS composition asdisclosed herein is a Microbial Surface Components Recognizing AdhesiveMatrix Molecule, or MSCRAMMs, which include, but are not limited to:EkeS, DsqA, KesK, KrkN, KrkN2, RkaS, RrkN, and KnkA. These MSCRAMMS aredescribed in WO 02/102829, which is hereby incorporated by reference.Additional MSCRAMMS, identified by GenBank Accession No., includeNP_373261.1, NP_373371.1, NP_374246.1, NP_374248.1, NP_374841.1,NP_374866.1, NP_375140.1, NP_375614.1, NP_375615.1, NP_375707.1,NP_375765.1, and NP_375773.1.

In certain aspects, a SA-MAPs composition can comprise a staphylococcalantigen selected from the group consisting of: FnBpA, FnBpB, LukD, LukE,LukF, SasA, SasD, SasG, SasI, SasK, SpA (and variants thereof), Eap,Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA,ClfB, Coa, Hla (e.g., H35 mutants), IsdC, SasF, vWbp, vWh andimmunogenic fragments thereof.

Some exemplary alternative SA antigens for use in the SA-MAPSimmunogenic composition as disclosed herein are discussed below.

Serine-Aspirate Repeat Protein E (SdrE)

The sdr genes are closely linked and tandemly arrayed, accordingly anyone of the Sdr proteins (e.g., SdrC, SdrD, SdrE, ClfA, and ClfB) can beused in the SA-MAPS immunogenic composition as disclosed herein. The Sdrproteins characteristically comprise an A region where there is highlyconserved amino acid sequence that can be used to derive a consensusTYTFTDYVD (SEQ ID NO: 20) motif. The motif exhibits slight variationbetween the different proteins. This variation, along with the consensussequence of the motif is described in U.S. Pat. No. 6,680,195. In theClf-Sdr proteins, this motif is highly conserved. The motif can be usedin immunogenic compositions to impart broad spectrum immunity tobacterial infections, and also can be used as an antigen in theproduction of monoclonal or polyclonal antibodies. Such an antibody canbe used to impart broad spectrum passive immunity.

The Sdr proteins differ from ClfA and ClfB by having two to fiveadditional 110-113 residue repeated sequences (B-motifs) located betweenregion A and the R-region. Each B-motif contains a consensusCa2+-binding EF-hand loop normally found in eukaryotic proteins. Thestructural integrity of a recombinant protein comprising the fiveB-repeats of SdrD was shown by bisANS fluorescence analysis to beCa2+-dependent, suggesting that the EF-hands are functional. When Ca2+was removed the structure collapsed to an unfolded conformation. Theoriginal structure was restored by addition of Ca2+. The C-terminalR-domains of the Sdr proteins contain 132-170 SD residues. These arefollowed by conserved wall-anchoring regions characteristic of manysurface proteins of Gram positive bacteria.

In the Sdr and Clf proteins this B motif is highly conserved while adegenerate version occurs in fibronectin binding MSCRAMMS, as well asthe collagen binding protein Cna. The B motifs, in conjunction with theR regions, are necessary for displaying the ligand-binding domain atsome distance from the cell surface. The repeated B motifs are onecommon denominator of the sub-group of SD repeat proteins describedherein. These motifs are found in different numbers in the three Sdrproteins from strain PFESA0237. There are clear distinctions between theindividual B motifs. The most conserved units are those located adjacentto the R regions (SdrC B2, SdrD B5 and SdrE B3). They differ from therest at several sites, especially in the C-terminal half A noteworthystructural detail is that adjacent B repeats are always separated by aproline residue present in the C-terminal region, but a proline neveroccurs between the last B repeats and the R region. Instead this linkeris characterized by a short acidic stretch. These differences areevidence that the end units have a different structural or functionalrole compared to the other B motifs. The N-terminal B motifs of SdrD andSdrE have drifted apart from the others, and there are numerous aminoacid alterations, including small insertions and deletions whereas theremaining internal B motifs are more highly conserved. Note that each ofthe three Sdr proteins has at least one B motif of each kind.

The C-terminal R-domains of the Sdr proteins contain 132-170 SDresidues. These are followed by conserved wall-anchoring regionscharacteristic of many surface proteins of Gram positive bacteria.

In some embodiments, a SdrE antigen can be used in the SA-MAPSimmunogenic composition as disclosed herein, and can comprises apolypeptide or peptide comprising at least part of SEQ ID NO: 8, whichcorresponds to the full length SdrE mature protein from S. aureus strainUSA300 (without the signal sequence).

(SEQ ID NO: 8) AENTSTENAKQDDATTSDNKEVVSETENNSTTENNSTNPIKKETNTDSQPEAKKESTSSSTQKQQNNVTATTETKPQNIEKENVKPSTDKTATEDTSVILEEKKAPNNTNNDVTTKPSTSEPSTSEIQTKPTTPQESTNIENSQPQPTPSKVDNQVTDATNPKEPVNVSKEELKNNPEKLKELVRNDSNTDHSTKPVATAPTSVAPKRVNAKMRFAVAQPAAVASNNVNDLIKVTKQTIKVGDGKDNVAAAHDGKDIEYDTEFTIDNKVKKGDTMTINYDKNVIPSDLTDKNDPIDITDPSGEVIAKGTFDKATKQITYTFTDYVDKYEDIKSRLTLYSYIDKKTVPNETSLNLTFATAGKETSQNVTVDYQDPMVHGDSNIQSIFTKLDEDKQTIEQQIYVNPLKKSATNTKVDIAGSQVDDYGNIKLGNGSTIIDQNTEIKVYKVNSDQQLPQSNRIYDFSQYEDVTSQFDNKKSFSNNVATLDFGDINSAYIIKVVSKYTPTSDGELDIAQGTSMRTTDKYGYYNYAGYSNFIVTSNDTGGGDGTVKPEEKLYKIGDYVWEDVDKDGVQGTDSKEKPMANVLVTLTYPDGTTKSVRTDANGHYEFGGLKDGETYTVKFETPTGYLPTKVNGTTDGEKDSNGSSVTVKINGKDDMSLDTGFYKEPKYNLGDYVWEDTNKDGIQDANEPGIKDVKVTLKDSTGKVIGTTTTDASGKYKFTDLDNGNYTVEFETPAGYTPTVKNTTADDKDSNGLTTTGVIKDADNMTLDSGFYKTPKYSLGDYVWYDSNKDGKQDSTEKGIKDVTVTLQNEKGEVIGTTKTDENGKYRFDNLDSGKYKVIFEKPAGLTQTVTNTTEDDKDADGGEVDVTITDHDDFTLDNGYFEEDTSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDAGKHTPVKPMSTTKDHHNKAKALPET

In some embodiments, a SA-MAPS composition can include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a SdrE of SEQ ID NO: 8. Incertain aspects a SdrE antigen peptide or polypeptide will have all, orpart of the amino acid sequence of SEQ ID NO: 8, e.g., will comprise atleast 50, or at least 60, or at least 70, or at least 80, or at least90, or at least 100, or at least 120, or at least 140, or at least 160,or at least 180, or at least 200, or at least 220 or at least 240 aminoacids of SEQ ID NO: 8. In one embodiment, a SdrD antigen peptide orpolypeptide present in the SA-MAPS immunogenic composition is apolypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to SEQ ID NO:8.

The term “SdrE protein” refers to a protein that includes isolatedwild-type SdrE polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SdrE proteins.

The term “SdrC protein” refers to a protein that includes isolatedwild-type SdrC polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SdrC proteins.

LukD, LukE, LukF

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to an LukD protein. Incertain aspects the LukD protein will have all or part of the amino acidsequence of accession number CAA73668/GI:2765304. The term “LukDprotein” refers to a protein that includes isolated wild-type LukDpolypeptides from staphylococcus bacteria and segments thereof, as wellas variants that stimulate an immune response against staphylococcusbacteria LukD proteins.

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to an LukE protein. Incertain aspects the LukE protein will have all or part of the amino acidsequence of accession number CAA73667.1/GI:2765303. The term “LukEprotein” refers to a protein that includes isolated wild-type LukEpolypeptides from staphylococcus bacteria and segments thereof, as wellas variants that stimulate an immune response against staphylococcusbacteria LukE proteins.

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to an LukF protein. Incertain aspects the LukF protein will have all or part of the amino acidsequence of accession number AAC60446.1/GI:410007. The term “LukFprotein” refers to a protein that includes isolated wild-type LukFpolypeptides from staphylococcus bacteria and segments thereof, as wellas variants that stimulate an immune response against staphylococcusbacteria LukF proteins.

S. aureus MntC/SitC/Saliva Binding Protein

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a MntC/SitC/Saliva BindingProtein. MntC/SitC/Saliva Binding Protein is an ABC transporter proteinand has homologues in S. epidermidis and S. aureus. It is referred toherein as MntC. This protein is a 32 kDa lipoprotein and is located inthe bacterial cell wall. See Sellman et al., and Cockayne et al.,Infect. Immun. 66: 3767 (1998). In S. epidermidis, it is a component ofan iron-regulated operon. It shows considerable homology to bothadhesins including FimA of S. parasanguis, and with lipoproteins of afamily of ABC transporters with proven or putative metal iron transportfunctions. The S. aureus homologue of MntC is known as saliva bindingprotein and was disclosed in U.S. Pat. No. 5,801,234, which isincorporated herein in its entirety by reference. The protein sequencefor the S. aureus homologue of MntC/SitC/Saliva Binding Protein is foundin GenBank accession number NP_371155 for strain Mu50, (also known asSAV0631), where the accession number for the nucleotide sequence for thecomplete genome of strain Mu50 is NC_002758.2 (coordinates704988-705917).

In alternative embodiments, a MntC antigen for use in the SA-MAPSimmunogenic composition as disclosed herein is a protein or peptidehaving an amino acid sequence of one the MntC proteins disclosed inTable 12 of U.S. Pat. No. 8,568,735, which is incorporated herein in itsentirety by reference. Other MntC antigens not identified herein aredisclosed encompassed for use in the SA-MAPS immunogenic composition,provided they are antigenic.

S. epidermidis SitC Protein

In one embodiment, a SA-MAPS composition may include a polypeptide,peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical or similar to a SitC protein. SitC isthe S. epidermidis homologue of MntC/SitC/Saliva Binding Protein and wasdisclosed in Sellman et al. (Sellman et al., Infect. Immun. 2005October; 73(10): 6591-6600). The protein sequence for SitC is found inGenBank accession number YP_1187886.1 (also known as SERP0290) and isdisclosed as SEQ ID NO: 121 in U.S. Pat. No. 8,568,735, which isincorporated herein in its entirety by reference. The accession numberfor the nucleotide sequence for the complete genome of strain RP62A, isNC_002976 (coordinates 293030-293959). Other candidate SitC moleculesmay be derived from various species of organisms for use in animmunogenic composition of the invention, some of which include, but arenot limited to: all or part of the amino acid sequence of accessionnumber BAE03450.1 (S. haemolyticus, JCSC1435 strain), AA004002.1 (S.epidermidis, strain ATCC 12228); BAE19233.1 (S. saprophyticus, strainATCC 15305); ABR57162.1 (S. xylosus. strain DSM20267); CAL27186.1 (S.carnosus, strain TM300).

The term “FnBpA protein” refers to a protein that includes isolatedwild-type FnBpA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria FnBpA proteins.

The term “FnBpB protein” refers to a protein that includes isolatedwild-type FnBpB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria FnBpB proteins.

The term “SasA protein” refers to a protein that includes isolatedwild-type SasA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasA proteins.

The term “SasD protein” refers to a protein that includes isolatedwild-type SasD polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasD proteins.

The term “SasG protein” refers to a protein that includes isolatedwild-type SasG polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasG proteins.

The term “SasI protein” refers to a protein that includes isolatedwild-type SasI polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasI proteins.

The term “SasK protein” refers to a protein that includes isolatedwild-type SasK polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasK proteins.

The term “EsxA protein” refers to a protein that includes isolatedwild-type EsxA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsxA proteins.

The term “EsxB protein” refers to a protein that includes isolatedwild-type EsxB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsxB proteins.

The term “Eap protein” refers to a protein that includes isolatedwild-type Eap polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Eap proteins.

The term “Ebh protein” refers to a protein that includes isolatedwild-type Ebh polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Ebh proteins.

The term “Emp protein” refers to a protein that includes isolatedwild-type Emp polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Emp proteins.

The term “EsaB protein” refers to a protein that includes isolatedwild-type EsaB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsaB proteins.

The term “EsaC protein” refers to a protein that includes isolatedwild-type EsaC polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsaC proteins.

The term “Coa protein” refers to a protein that includes isolatedwild-type Coa polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Coa proteins.

The term “SasF protein” refers to a protein that includes isolatedwild-type SasF polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasF proteins.

The term “vWbp protein” refers to a protein that includes isolatedwild-type vWbp (von Willebrand factor binding protein) polypeptides fromstaphylococcus bacteria and segments thereof, as well as variants thatstimulate an immune response against staphylococcus bacteria vWbpproteins.

The term “vWh protein” refers to a protein that includes isolatedwild-type vWh (von Willebrand factor binding protein homolog)polypeptides from staphylococcus bacteria and segments thereof, as wellas variants that stimulate an immune response against staphylococcusbacteria vWh proteins.

In certain embodiments, the claimed invention specifically excludes 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more of FnBpA, FnBpB, LukD (GI:2765304),LukE (GI:2765303), LukF (GI:12231006), SasA, SasD, SasG, SasI, SasK, SpA(and variants thereof), Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC,SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla (e.g., H35 mutants), IsdC,SasF, vWbp, vWh, 52 kDa vitronectin binding protein (WO 01/60852), Aaa(GenBank CAC80837), Aap (GenBank accession AJ249487), Ant (GenBankaccession NP-372518), autolysin glucosaminidase, autolysin amidase, Cna,collagen binding protein (U.S. Pat. No. 6,288,214), EFB (FIB), Elastinbinding protein (EbpS), EPB, FbpA, fibrinogen binding protein (U.S. Pat.No. 6,008,341), Fibronectin binding protein (U.S. Pat. No. 5,840,846),FnbA, FnbB, GehD (US 2002/0169288), HarA, HBP, Immunodominant ABCtransporter, IsaA/P isA, laminin receptor, Lipase GehD, MAP, Mg2+transporter, MHC II analogue (U.S. Pat. No. 5,648,240), MRPII, Npase,RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI,SdrF (WO 00/12689), SdrG/Fig (WO 00/12689), SdrH (WO 00/12689), SEAexotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABCtransporter, SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234),SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein (see PCTpublications WO2007/113222, WO2007/113223, WO2006/032472, WO2006/032475,WO2006/032500, each of which is incorporated herein by reference intheir entirety). In certain aspects, the bacterial antigen is astaphylococcal antigen. The staphylococcal antigen can be selected fromthe group consisting of: FnBpA, FnBpB, LukD, LukE, LukF, SasA, SasD,SasG, SasI, SasK, SpA (and variants thereof), Eap, Ebh, Emp, EsaB, EsaC,EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla (e.g.,H35 mutants), IsdC, SasF, vWbp, vWh and immunogenic fragments thereof.Certain embodiments are directed to an immunogenic compositioncomprising an isolated Protein A (SpA) specific antibody and a bacterialantigen, wherein the Protein A specific antibody enhances an immuneresponse to the bacterial antigen. In certain aspects, the antibody is apolyclonal antibody, a monoclonal antibody, or an antibody fragment. Instill further aspects, the bacterial antigen is comprised in or on abacterium. The bacteria can be attenuated bacteria, in particularattenuated staphylococcal bacteria.

In certain embodiments a subject is administered a SA-MAPS compositioncomprising a SA antigen, wherein the SA antigen is Hla209 or any SAantigen selected from any of: FnBpA antigen or immunogenic fragmentthereof, FnBpB antigen or immunogenic fragment thereof, LukD antigen orimmunogenic fragment thereof, LukE antigen or immunogenic fragmentthereof, LukF antigen or immunogenic fragment thereof, SasA antigen orimmunogenic fragment thereof, SasD antigen or immunogenic fragmentthereof, SasG antigen or immunogenic fragment thereof, SasI antigen orimmunogenic fragment thereof, SasK antigen or immunogenic fragmentthereof, SpA (and variants thereof) antigen or immunogenic fragmentthereof, Eap antigen or immunogenic fragment thereof, Ebh antigen orimmunogenic fragment thereof, Emp antigen or immunogenic fragmentthereof, EsaB antigen or immunogenic fragment thereof, EsaC antigen orimmunogenic fragment thereof, EsxA antigen or immunogenic fragmentthereof, EsxB antigen or immunogenic fragment thereof, SdrC antigen orimmunogenic fragment thereof, SdrD antigen or immunogenic fragmentthereof, SdrE antigen or immunogenic fragment thereof, IsdA antigen orimmunogenic fragment thereof, IsdB antigen or immunogenic fragmentthereof, ClfA antigen or immunogenic fragment thereof, ClfB antigen orimmunogenic fragment thereof, Coa antigen or immunogenic fragmentthereof, Hla (e.g., H35 mutants) antigen or immunogenic fragmentthereof, IsdC antigen or immunogenic fragment thereof, SasF antigen orimmunogenic fragment thereof, vWbp antigen or immunogenic fragmentthereof, vWh antigen or immunogenic fragment thereof.

Combinations of SA Antigens Present on the SA-MAPS ImmunogenicComposition

In some embodiments, a SA-MAPS complex comprises at least 2 SA antigens,e.g., Hla, such as but not limited to Hla (209) as disclosed herein, andone or more SA antigens selected from a Clumping factor A (ClfA),Clumping factor B (ClfB), serine-aspirate repeat protein D (SdrD),serine-aspirate repeat protein E (SdrE), Iron regulator surface proteinA (IsdA), Iron regulator surface protein B (IsdB), Leukotoxin D (LukD),or Leukotoxin E (LukE), or fragments thereof.

In some embodiments, a SA-MAPS immunogenic composition as disclosedherein can comprise all 6 SA antigens selected from: hemolysin (HI)(e.g., hemolysin α or Hla209), Clumping factor A (ClfA), Clumping factorB (ClfB), serine-aspirate repeat protein D (SdrD), Iron regulatorsurface protein A (IsdA) and Iron regulator surface protein B (IsdB), orfragments thereof, for example, but not limited to: Hla209(27-319), ClfA(221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447)or proteins or peptides having at least 85% sequence identity thereto.It is envisioned that any of the above listed SA antigens can besubstituted for a different SA peptide or polypeptide antigen known toone of ordinary skill in the art.

In alternative embodiments, the SA-MAPS immunogenic compositions asdisclosed herein can comprise any SA antigen that elicits an immuneresponse in a subject. In some embodiments, the SA-MAPS compositioncomprises at least one, or at least 2 SA antigens. In some embodiments,the SA-MAPS immunogenic composition comprises at least 2, or at least 3,or at least 4, or between 2-4, or between 3-5, or between 6-8, orbetween 8-10 or between 10-12, or between 10-15, or between 15-20 ormore than 20 SA protein or polypeptide antigens. In some embodiments,the antigens can be the same, e.g., all ClfA antigens, or a combinationof different antigens, e.g., Hla209, ClfA, ClfB etc. In someembodiments, the SA-MAPS composition comprises at least a Hla209 antigen(e.g., Hla209(27-319)) and at least 1 more, or at least 2 more, or atleast 3 more or at least 4 more, or at least 5 more SA antigens asdisclosed herein.

Exemplary combinations of different SA antigen present on a SA-MAPSimmunogenic composition as disclosed herein are shown in Tables 3A-3G.

In particular, Tables 3A-3G show exemplary SA antigens present onSA-MAPS complexes which are useful in the compositions and methods asdisclosed herein. Tables 3A-3G have used an exemplary set of 9 SAantigens, and it is envisioned that any of the SA antigens can besubstituted for a different SA peptide or polypeptide antigen known toone of ordinary skill in the art. In some embodiments, a SA-MAPSimmunogenic composition comprises a combination of 2, 3, 4, 5 or 6 ofthe exemplary SA antigens selected from hemolysin (HI) (e.g., hemolysinα or Hla209), Clumping factor A (ClfA), Clumping factor B (ClfB),serine-aspirate repeat protein D (SdrD), Iron regulator surface proteinA (IsdA) and Iron regulator surface protein B (IsdB), or fragmentsthereof, e.g., Hla209(27-319), ClfA (221-559), ClfB (203-542), SdrD(246-682), IsdA (47-324), IsdB (48-447) or proteins or peptides havingat least 85% sequence identity thereto. It is noted that reference toLUKD, LUKE and SDRE in Tables 3A-3G are examples of other SA antigensthat can be used in place of (i.e., substituted) any of, or in additionto, the exemplary Hla209(27-319), ClfA (221-559), ClfB (203-542), SdrD(246-682), IsdA (47-324), IsdB (48-447) SA antigens.

Table 3B-3G show exemplary combinations of 2, 3, 4, 5, 6, 7, 8 and 9antigens present in the MAPS complex. HLA209=Hla (209), CLFA=ClfA(221-559), CLFB=ClfB (203-542), SDRD=SdrD (246-682), SDRE=SdrE,ISDA=IsdA (47-324), ISDB=IsdB (48447), LUKD=LukD, LUKE=LukE.

TABLE 3A SA-MAPS with at least 1 SA-antigens (9 combinations) HLA209CLFA CLFB SDRD ISDA ISDB SDRE LUKD LUKE

TABLE 3B SA-MAPS with different combinations of 2 SA-antigens (24combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA CLFB SDRD ISDA ISDB SDRE LUKD LUKE CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFB, CLFB SDRD ISDA ISDB SDRE LUKD LUKE SDRD CLFB,CLFB, CLFB, CLFB, CLFB, SDRD, SDRD, SDRD, ISDA ISDB SDRE LUKD LUKE ISDAISDB SDRE SDRD, SDRD, ISDA, ISDA, ISDA, ISDA, ISDB, ISDB, LUKD LUKE ISDBSDRE LUKD LUKE SDRE LUKD ISDB, SDRE, SDRE, LUKD, LUKE LUKD LUKE LUKE

TABLE 3C SA-MAPS with different combinations of 3 SA-antigens (84combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFB CLFA, SDRD CLFA, ISDA CLFA, ISDB CLFA, SDRE CLFA,LUKD CLFA, LUKE CLFB, SDRD HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, S HLA209, HLA209, CLFB, ISDA CLFB, ISDB CLFB, SDRE CLFB, LUKDCLFB, LUKE DRD, ISDA SDRD, ISDB SDRD, SDRE HLA209, HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, SDRD, LUKD SDRD, LUKE ISDA, ISDBISDA, SDRE ISDA, LUKD ISDA, LUKE ISDB, SDRE ISDB, LUKD HLA209, HLA209,HLA209, HLA209, CLFA, CLFA, CLFA, CLFA, ISDB, LUKE SDRE, LUKD SDRE, LUKELUKD, LUKE CLFB, SDRD CLFB, ISDA CLFB, ISDB CLFB, SDRE CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, LUKD CLFB, LUKE SDRD, ISDA SDRD,ISDB SDRD, SDRE SDRD, LUKD SDRD, LUKE ISDA, ISDB CLFA, ISDA, CLFA, ISDA,CLFA, ISDA, CLFA, CLFA, ISDB, CLFA, ISDB, CLFA, SDRE, CLFA, SDRE, SDRELUKD LUKE ISDB, SDRE LUKD LUKE LUKD LUKE CLFA, CLFB, SDRD, CLFB, SDRD,CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, ISDA, CLFB, ISDA, LUKD, LUKEISDA ISDB SDRE LUKD LUKE ISDB SDRE CLFB, ISDA, CLFB, ISDA, CLFB, ISDB,CLFB, ISDB, CLFB, ISDB, CLFB, SDRE, CLFB, SDRE, CLFB, LUKD, LUKD LUKESDRE LUKD LUKE LUKD LUKE LUKE SDRD, SDRD, SDRD, SDRD, SDRD, SDRD, SDRD,SDRD, ISDA, ISDB ISDA, SDRE ISDA, LUKD ISDA, LUKE ISDB, SDRE ISDB, LUKDISDB, LUKE SDRE, LUKD SDRD, SDRD, ISDA, ISDA, ISDA, ISDA, ISDA, ISDA,SDRE, LUKE LUKD, LUKE ISDB, SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE,LUKE LUKD, LUKE ISDB, ISDB, ISDB, SDRE, SDRE, LUKD SDRE, LUKE LUKD, LUKELUKD, LUKE

TABLE 3D SA-MAPS with different combinations of 4 SA-antigens (126combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, SDRD CLFB,ISDA CLFB, ISDB CLFB, SDRE CLFB, LUKD CLFB, LUKE SDRD, ISDA SDRD, ISDBHLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFA,CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, SDRD, SDRE SDRD, LUKD SDRD,LUKE ISDA, ISDB ISDA, SDRE ISDA, LUKD ISDA, LUKE ISDB, SDRE HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFA, CLFA,CLFA, CLFA, CLFA, CLFB, CLFB, CLFB, ISDB, LUKD ISDB, LUKE RSDE, LUKDSDRE, LUKE LUKD, LUKE SDRD, ISDA SDRD, ISDB SDRD, SDRE HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFB, CLFB, CLFB, CLFB,CLFB, CLFB, CLFB, CLFB, SDRD, LUKD SDRD, LUKE ISDA, ISDB ISDA, SDREISDA, LUKD ISDA, LUKE ISDB, SDRE ISDB, LUKD HLA209, HLA209, HLA209,HLA209, HLA209, S HLA209, S HLA209, HLA209, S CLFB, CLFB, CLFB, CLFB,DRD, DRD, SDRD, DRD, ISDB, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE ISDA,ISDB ISDA, SDRE ISDA, LUKD ISDA, LUKE HLA209, HLA209, HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, SDRD, ISDB, SDRD, ISDB, SDRD, ISDB,SDRD, SDRE, SDRD, SDRE, SDRD, LUKD, ISDA, ISDB, ISDA, ISDB, SDRE LUKDLUKE LUKD LUKE LUKE SDRE LUKD HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, HLA209, HLA209, ISDA, ISDB, ISDA, SDRE, ISDA, SDRE, ISDA, LUKD,ISDB, SDRE, ISDB, SDRE, ISDB, LUKD, SDRE, LUKD, LUKE LUKD LUKE LUKE LUKDLUKE LUKE LUKE CLFA, CLFB, CLFA, CLFB, CLFA, CLFB, CLFA, CLFB, CLFA,CLFB, CLFA, CLFB, CLFA, CLFA, SDRD, SDRD, SDRD SDRD, SDRD, ISDA, CLFB,ISDA, CLFB, ISDA, ISDA ISDB SDRE LUKD LUKE ISDB SDRE LUKD CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, ISDA, CLFB, ISDB, CLFB, ISDB,CLFB, ISDB, CLFB, SDRE, CLFB, SDRE, CLFB, LUKD, SDRD, ISDA, LUKE SDRELUKD LUKE LUKD LUKE LUKE ISDB CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA,CLFA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDB, SDRD, ISDB, SDRD,ISDB, SDRD, SDRE, SDRD, SDRE, SDRE LUKD LUKE SDRE LUKD LUKE LUKD LUKECLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, ISDA, CLFA, SDRD, LUK ISDA,ISDB, ISDA, ISDB, ISDA, ISDB, ISDA, SDRE, ISDA, SDRE, LUKD, ISDB, SDRE,D, LUKE SDRE LUKD LUKE LUKD LUKE LUKE LUKD CLFA, CLFA, CLFA, CLFB, CLFB,CLFB, CLFB, CLFB, ISDB, SDRE, ISDB, LUKD, SDRE, LUKD, SDRD, ISDA, SDRD,ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDB, LUKE LUKE LUKE ISDB SDRE LUKDLUKE SDRE CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, ISDA, CLFB, CLFB, SDRD,ISDB, SDRD, ISDB, SDRD, SDRE, SDRD, SDRE, SDRD, ISDB, ISDA, ISDB, ISDA,ISDB, LUKD LUKE LUKD LUKE LUKD, LUKE SDRE LUKD LUKE CLFB, CLFB, CLFB,CLFB, CLFB, CLFB, CLFB, SDRD, ISDA, SDRE, ISDA, SDRE, ISDA, LUKD, ISDB,SDRE, ISDB, SDRE, ISDB, LUKD, SDRE, LUKD, ISDA, ISDB, LUKD LUKE LUKELUKD LUKE LUKE LUKE SDRE SDRD, SDRD, SDRD, SDRD, SDRD, SDRD, SDRD, SDRD,ISDA, ISDB, ISDA, ISDB, ISDA, SDRE, ISDA, SDRE, ISDA, LUK ISDB, SDRE,ISDB, SDRE, ISDB, LUKD, LUKD LUKE LUKD LUKE D, LUKE LUKD LUKE LUKE SDRD,ISDA, ISDA, ISDB, ISDA, ISDB, ISDA, SDRE, ISDB, SDRE, SDRE, LUKD, ISDB,SDRE, SDRE, L LUKD, LUKD, LUKD, LUKE LUKD UKE LUKE LUKE LUKE

TABLE 3E SA-MAPS with different combinations of 5 SA-antigens (126combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, SDRD,CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, ISDA, CLFB, ISDA,CLFB, ISDA, ISDA ISDB SDRE LUKD LUKE ISDB SDRE LUKD HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFB, ISDA, CLFB, ISDB, CLFB, ISDB, CLFB, ISDB,CLFB, SDRE, CLFB, SDRE, CLFB, LUKD, SDRD, ISDA, LUKE SDRE LUKD LUKE LUKDLUKE LUKE ISDB HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, SDRD, ISDA,SDRD, ISDA, SDRD, ISDA, SDRD, ISDB, SDRD, ISDB, SDRD, ISDB, SDRD, SDRE,SDRD, SDRE, SDRE LUKD LUKE SDRE LUKD LUKE LUKD LUKE HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, SDRD, LUKD, ISDA, ISDB, ISDA, ISDB, ISDA, ISDB,ISDA, SDRE, ISDA, SDRE, ISDA, LUKD, ISDB, SDRE, LUKE SDRE LUKD LUKE LUKDLUKE LUKE LUKD HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFB, CLFB, CLFB, CLFB, CLFB, ISDB, SDRE,ISDB, SDRE, LUKD, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD,ISDB, LUKE LUKD, LUKE LUKE ISDB SDRE LUKD LUKE SDRE HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFB, CLFB, CLFB, CLFB,CLFB, CLFB, CLFB, CLFB, SDRD, ISDB, SDRD, ISDB, SDRD, SDRE, SDRD, SDRE,SDRD, LUKD, ISDA, ISDB, ISDA, ISDB, ISDA, ISDB, LUKD LUKE LUKD LUKE LUKESDRE LUKD LUKE HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFB, CLFB, CLFB, CLFB, CLFB, ISDB, CLFB, CLFB, SDRD, ISDA,SDRE, ISDA, SDRE, ISDA, LUKD, ISDB, SDRE, SDRE, ISDB, LUKD, SDRE, LUKD,ISDA, ISDB, LUKD LUKE LUKE LUKD LUKE LUKE LUKE SDRE HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, SDRD, ISDA, SDRD, SDRD,ISD SDRD, SDRD, ISDA, SDRD, SDRD, ISD SDRD, ISDB, ISDB, ISDA, ISDB, A,SDRE, ISDA, SDRE, LUKD, ISDB, SDRE, B, SDRE, LUKD, LUKD LUKE LUKD LUKELUKE LUKD LUKE LUKE HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,CLFA, CLFA, SDRD, ISDA, ISDA, ISD ISDA, ISDA, SDRE, ISDB, SDRE, CLFB,SDRD, CLFB, SDRD, SDRE, LUKD, ISDB, SDRE, B, SDRE, ISDB, LUKD, LUKD,LUKD, ISDA, ISDB ISDA, SDRE LUKE LUKD LUKE LUKE LUKE LUKE CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD,CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, ISDA, LUKDISDA, LUKE ISDB, SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE, LUKE LUKD,LUKE CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, ISDA, CLFB,ISDA, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA, CLFB, ISDB, CLFB,ISDB, ISDB, SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE, LUKE LUKD, SDRE,LUKD SDRE, LUKE CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB,ISDB, CLFB, SDRE, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD,ISDA, SDRD, ISDA, LUKD, LUKE LUKD, LUKE ISDB, SDRE ISDB, LUKD ISDB, LUKESDRE, LUKD SDRE, LUKE LUKD, LUKE CLFA, CLFA, CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, SDRD, ISDB, SDRD, ISDB, SDRD, ISDB, SDRD, SDRE, ISDA, ISDB,ISDA, ISDB, ISDA, ISDB, ISDA, SDRE, SDRE, LUKD SDRE, LUKE LUKD, LUKELUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE LUKD, LUKE CLFA, CLFB, CLFB,CLFB, CLFB, CLFB, CLFB, CLFB, ISDB, SDRE, SDRD, ISDA, SDRD, ISDA, SDRD,ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDB, LUKD, LUKE ISDB,SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE SDRE, LUKDCLFB, CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, ISDA, CLFB, SDRD, ISDB, SDRD,ISDB, SDRD, SDRE, ISDA, ISDB, ISDA, ISDB, ISDA, ISDB, SDRE, ISDB, SDRE,SDRE, LUKE LUKD, LUKE LUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE LUKD,LUKE LUKD, LUKE SDRD, SDRD, SDRD, SDRD, SDRD, ISDA, ISDA, ISDB, ISDA,ISDB, ISDA, ISDB, ISDA, SDRE, ISDB, SDRE, ISDB, SDRE, SDRE, LUKD SDRE,LUKE LUKD, LUKE LUKD, LUKE LUKD, LUKE LUKD, LUKE

TABLE 3F SA-MAPS with different combinations of 6 SA-antigens (84combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, SDRD,CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD,CLFB, SDRD, ISDA, ISDB ISDA, SDRE ISDA, LUKD ISDA, LUKE ISDB, SDRE ISDB,LUKD ISDB, LUKE SDRE, LUKD HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, HLA209, HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA,CLFB, SDRD, CLFB, SDRD, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA,CLFB, ISDA, CLFB, ISDA, SDRE, LUKE LUKD, LUKE ISDB, SDRE ISDB, LUKDISDB, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE HLA209, HLA209, HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, CLFA, CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFB, ISDB, CLFB, ISDB, CLFB, SDRE, SDRD, ISDA, SDRD,ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRE, LUKD LUKD, LUKE LUKD,LUKE ISDB, SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE, LUKE HLA209,HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, SDRD, ISDA, SDRD, ISDB, SDRD, ISDB,SDRD, ISDB, SDRD, SDRE, ISDA, ISDB, ISDA, ISDB, ISDA, ISDB, LUKD, LUKESDRE, LUKD SDRE, LUKE LUKD, LUKE LUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD,LUKE HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,CLFA, CLFA, CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, ISDA, SDRE, ISDB, SDRE,SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA,LUKD, LUKE LUKD, LUKE ISDB, SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE,LUKE LUKD, LUKE HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, SDRD, ISDB,SDRD, ISDB, SDRD, ISDB, SDRD, SDRE, ISDA, ISDB, ISDA, ISDB, ISDA, ISDB,ISDA, SDRE, SDRE, LUKD SDRE, LUKE LUKD, LUKE LUKD, LUKE SDRE, LUKD SDRE,LUKE LUKD, LUKE LUKD, LUKE HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, HLA209, CLFA, CLFB, SDRD, SDRD, ISDA, SDRD, ISDA, SDRD, SDRD,ISDA, ISD CLFB, SDRD, ISDB, SDRE, ISDA, ISDB, ISDB, ISDB, ISDA, SDRE,ISDB, SDRE, B, SDRE, ISDA, LUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKELUKD, LUKE LUKD, LUKE LUKD, LUKE ISDB, SDRE CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD,CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, ISDA, ISDA, ISDB, ISDA,ISDA, ISDA, ISDB, ISDB, ISDB, ISDB, LUKD LUKE SDRE, LUKD SDRE, LUKELUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE CLFA, CLFA, CLFA, CLFA,CLFA, CLFA, CLFA, CLFA, CLFB, SDRD, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA,CLFB, ISDA, CLFB, ISDB, SDRD, ISDA, SDRD, ISDA, SDRE, ISDB, SDRE, ISDB,ISDB, LUKD, SDRE, SDRE, ISDB, ISDB, LUKD, LUKE LUKD SDRE, LUKE LUKELUKD, LUKE LUKD, LUKE SDRE, LUKD SDRE, LUKE CLFA, CLFA, CLFA, CLFA,CLFB, CLFB, CLFB, CLFB, SDRD, ISDA, SDRD, ISDA, SDRD, ISDB, ISDA, ISDB,SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA, ISDB, SDRE, SDRE, SDRE,ISDB, ISDB, ISDB, SDRE, LUKD, LUKE LUKD, LUKE LUKD, LUKE LUKD, LUKESDRE, LUKD SDRE, LUKE LUKD, LUKE LUKD, LUKE CLFB, CLFB, SDRD, SDRD,ISDB, ISDA, ISDB, ISDA, ISDB, SDRE, SDRE, SDRE, LUKD, LUKE LUKD, LUKELUKD, LUKE

TABLE 3G SA-MAPS with different combinations of 7 SA-antigens (36combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, SDRD,CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD,CLFB, SDRD, ISDA, ISDB, ISDA, ISDA, ISDA, ISDA, ISDA, ISDB, SD ISDB,SDRE ISDB, LUKD ISDB, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE RE, LUKDSDRE, LUKE HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, SDRD,CLFB, SDRD, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA, CLFB, ISDA, CLFB, ISDB,SDRD, ISDA, ISDB, SDRE, ISDB, ISDB, ISDB, SDRE, SDRE, ISDB, LUKD, LUKELUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKE LUKD, LUKE LUKD, LUKE SDRE,LUKD HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, CLFB, CLFB, SDRD, ISDA, SDRD, ISDA,SDRD, ISDA, SDRD, ISDB, ISDA, ISDB, SDRD, ISDA, SDRD, ISDA, SDRD, ISDA,ISDB, ISDB, SDRE, SDRE, SDRE, ISDB, ISDB, ISDB, SDRE, LUKE LUKD, LUKELUKD, LUKE LUKD, LUKE LUKD, LUKE SDRE, LUKD SDRE, LUKE LUKD, LUKEHLA209, HLA209, HLA209, HLA209, CLFA, CLFA, CLFA, CLFA, CLFB, CLFB,CLFB, DSRD, ISDA, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, CLFB, SDRD, SDRD,ISDA, SDRD, ISDB, ISDA, ISDB, ISDB, ISDA, ISDA, ISDB, ISDA, ISDA, SDRE,SDRE, SDRE, LUKD, SDRE, LUKD, ISDB, SDRE, SDRE, ISDB, LUKD, SDRE, LUKDLUKD, LUKE LUKD, LUKE LUKE LUKE LUKD LUKE LUKE LUKE CLFA, CLFA, CLFA,CLFB, CLFB, SDRD, CLFB, ISDA, SDRD, ISDA, SDRD, ISDA, ISDB, ISDB, SDRE,ISDB, ISDB, SDRE, LUKD, LUKD, SDRE, LUKD, SDRE, LUKD, LUKE LUKE LUKELUKE

TABLE 3F SA-MAPS with different combinations of 8 SA-antigens (9combinations) HLA209, HLA209, HLA209, HLA209, HLA209, HLA209, HLA209,HLA209, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFA, CLFB, CLFB,CLFB, CLFB, CLFB, CLFB, CLFB, CLFB, SDRD, SDRD, SDRD, SDRD, ISDA, SDRD,SDRD, SDRD, SDRD, ISDA, ISDB, ISDA, ISDA, ISDA, ISDB, ISDB, ISDA, ISDB,ISDA, ISDB, ISDA, SDRE, ISDB, SDRE, SDRE, ISDB, SDRE, ISDB, SDRE, SDRE,SDRE, SDRE, LUKD, LUKD, LUKD, LUKD, LUKD, LUKD, LUKD, LUKD LUKE LUKELUKE LUKE LUKE LUKE LUKE LUKE

TABLE 3G an exemplary SA-MAPS with all 9 SA-antigensHLA209,CLFA,CLFB,SDRD,ISDA,ISDB,SDRE,LUKD,LUKE

It is envisioned that any of the above-identified antigens in Tables3A-3G can be switched out for a different SA antigen, including adifferent peptides or polypeptides of ClfA, ClfB, SdrD, SdrE, IsdA,IsdB, LukD, or LukE, or peptides or polypeptides at least 85% sequenceidentity thereto, or completely different SA antigens. In someembodiments, a SA antigen identified in tables 3A-3G can be substitutedor switched out with a non-SA antigen, as disclosed herein.

Accordingly, in some embodiments, an ordinary skilled artisan cansubstitute any of the antigens listed in Tables 3A-3G with any other SAantigen not listed herein and known to an ordinary skilled artisan, oreven substitute a SA antigen listed in Tables 3A-3G with a non-SAantigen.

In addition to one or more S. aureus antigens present in the MAPScomplex, the MAPS complex may comprise non-S. aureus (non-SA)immunogenic antigens, including but not limited to pathogenic peptides,toxins, toxoids, subunits thereof, or combinations thereof (e.g.,cholera toxin, tetanus toxoid).

In some embodiments, an antigen is derived (e.g., obtained) from apathogenic organism. In some embodiments, the antigen is a cancer ortumor antigen, e.g., an antigen derived from a tumor or cancer cell.

In some embodiments, an antigen derived from a pathogenic organism is anantigen associated with an infectious disease; it can be derived fromany of a variety of infectious agents, including virus, bacterium,fungus or parasite.

In some embodiments, a target antigen is any antigen associated with apathology, for example an infectious disease or pathogen, or cancer oran immune disease such as an autoimmune disease. In some embodiments, anantigen can be expressed by any of a variety of infectious agents,including virus, bacterium, fungus or parasite. A target antigen for usein the methods and compositions as disclosed herein can also include,for example, pathogenic peptides, toxins, toxoids, subunits thereof, orcombinations thereof (e.g., cholera toxin, tetanus toxoid).

Non-limiting examples of infectious viruses include: Retroviridae;Picornaviridae (for example, polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (such as strains that cause gastroenteritis); Togaviridae(for example, equine encephalitis viruses, rubella viruses); Flaviridae(for example, dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bungaviridae (forexample, Hantaan viruses, bunga viruses, phleboviruses and Nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.,reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), Marek's disease virus, herpes viruses);Poxviridae (variola viruses, vaccinia viruses, pox viruses); andridoviridae (such as African swine fever virus); and unclassifiedviruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses). The compositions and methods described herein arecontemplated for use in treating infections with these viral agents.

Examples of fungal infections that may be addressed by inclusion ofantigens in the present embodiments include aspergillosis; thrush(caused by Candida albicans); cryptococcosis (caused by Cryptococcus);and histoplasmosis. Thus, examples of infectious fungi include, but arenot limited to, Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,Candida albicans. Components of these organisms can be included asantigens in the MAPS described herein.

In one aspect of the invention, an non-SA antigen to be used incombination with one or more SA antigens on the MAPS complex is derivedfrom an infectious microbe such as Bordatella pertussis, Brucella,Enterococci sp., Neisseria meningitidis, Neisseria gonorrheae,Moraxella, typeable or nontypeable Haemophilus, Pseudomonas, Salmonella,Shigella, Enterobacter, Citrobacter, Klebsiella, E. coli, Helicobacterpylori, Clostridia, Bacteroides, Chlamydiaceae, Vibrio cholera,Mycoplasma, Treponemes, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellulare,M. kansaii, M. gordonae, M. leprae), Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus anthracis, Corynebacteriumdiphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Leptospira sps., Pasteurella multocida,Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis,Treponema pallidium, Treponema pertenue, and Actinomyces israelli.

In some embodiments, a non-SA antigen useful in a SA-MAPS complex asdisclosed herein is an antigen from an enteric bacterium, or non-entericgram-negative bacteria. In some embodiments, a non-SA antigen useful ina SA-MAPS complex as disclosed herein can be selected from any of, or acombination of: a pneumococcal antigen, tuberculous antigen, HIVantigen, seasonal or epidemic influenza antigen, pertussis antigen,meningococcal antigen, haemophilus antigen, HPV antigen, E. coliantigens, salmonella antigens, enterobacter antigens, acinetobacterpathogen antigens, pseudomona antigens, klebsiella antigens, citrobacterantigens, serratia antigens, Clostridium difficile antigens from anenteric bacteria, antigens from non-enteric gram-negative bacteria,toxoids, toxins or toxin portions thereof.

In some embodiments, a non-SA antigen useful in a SA-MAPS complex asdisclosed herein is a pneumococcal antigen, a tuberculosis antigen, ananthrax antigen, a HIV antigens, a seasonal or epidemic influenzaantigen, a HPV antigen, an Acinetobacter antigens, a-Clostridiumdifficile antigen, an enteric Gram-negative bacterial antigen ornonenteric Gram-negative bacterial antigen, a Gram-positive bacterialantigens, a toxoid, toxin or toxin portion, a fungal antigen, a viralantigen, a cancer antigen or any combinations thereof.

In some embodiments, a non-SA antigen useful in a SA-MAPS complex asdisclosed herein is an enteric Gram-negative bacterial antigen, selectedfrom the group of: E. coli antigens, Salmonella antigens, Enterobacterantigens, Klebsiella antigens, Citrobacter antigens and Serratiaantigens, or combinations thereof. In some embodiments, a non-SA antigenuseful in a SA-MAPS complex as disclosed herein is a nonentericGram-negative bacterial antigens are selected from the group of:Pertussis antigens, Meningococcal antigens, Haemophilus antigens, andPseudomonas antigens or combinations thereof.

Additional parasite pathogens from which antigens can be derivedinclude, for example: Entamoeba histolytica, Plasmodium falciparum,Leishmania sp., Toxoplasma gondii, Rickettsia, and the Helminths.

In some embodiments, a non-SA antigen useful in a SA-MAPS complex asdisclosed herein is a truncated pneumococcal PsaA protein, pneumolysintoxoid pneumococcal serine/threonine protein kinase (StkP), pneumococcalserine/threonine protein kinase repeating unit (StkPR), pneumococcalPcsB protein, staphylococcal alpha hemolysin, Mycobacterium tuberculosismtb protein ESAT-6, M. tuberculosis cell wall core antigen, ChlamydiaCT144, CT242 or CT812 polypeptides or fragments of these, Chlamydia DNAgyrase subunit B, Chlamydia sulfite synthesis/biphosphate phosphatase,Chlamydia cell division protein FtsY, Chlamydia methionyl-tRNAsynthetase, Chlamydia DNA helicase (uvrD), Chlamydia ATP synthasesubunit I (atpI), or Chlamydia metal dependent hydrolase.

In some embodiments, a non-SA antigen useful in a SA-MAPS complex asdisclosed herein is an antigen from Myocobacterium tuberculosis (TB).One example of a TB antigen is TbH9 (also known as Mtb 39A). Other TBantigens include, but are not limited to, DPV (also known as Mtb8.4),381, Mtb41, Mtb40, Mtb32A, Mtb64, Mtb83, Mtb9.9A, Mtb9.8, Mtb16, Mtb72f,Mtb59f, Mtb88f, Mtb7lf, Mtb46f and Mtb3lf, wherein “f” indicates that itis a fusion or two or more proteins.

In some embodiments, a non-SA antigen useful in a SA-MAPS complex asdisclosed herein can be derived from a Chlamydia species for use in theimmunogenic compositions of the present invention. Chlamydiaceae(consisting of Chlamydiae and Chlamydophila), are obligate intracellulargram-negative bacteria. Chlamydia trachomatis infections are among themost prevalent bacterial sexually transmitted infections, and perhaps 89million new cases of genital chlamydial infection occur each year. TheChlamydia of the present invention include, for example, C. trachomatis,Chlamydophila pneumoniae, C. muridarum, C. suis, Chlamydophila abortus,Chlamydophila psittaci, Chlamydophila caviae, Chlamydophila felis,Chlamydophila pecorum, and C. pneumoniae. Animal models of chlamydialinfection have established that T-cells play a critical role both in theclearance of the initial infection and in protection from re-infectionof susceptible hosts. Hence, the immunogenic compositions as disclosedherein can be used to provide particular value by eliciting cellularimmune responses against chlamydial infection.

More specifically, Chlamydial antigens useful as a non-SA antigen in aSA-MAPS complex as disclosed herein include DNA gyrase subunit B,sulfite synthesis/biphosphate phosphatase, cell division protein FtsY,methionyl-tRNA synthetase, DNA helicase (uvrD); ATP synthase subunit I(atpI) or a metal-dependent hydrolase (U.S. Patent Application Pub. No.20090028891). Additional Chlamyidia trachomatis antigens include CT144polypeptide, a peptide having amino acid residues 67-86 of CT144, apeptide having amino acid residues 77-96 of CT144, CT242 protein, apeptide having amino acids 109-117 of CT242, a peptide having aminoacids 112-120 of CT242 polypeptide, CT812 protein (from the pmpD gene),a peptide having amino acid residues 103-111 of the CT812 protein; andseveral other antigenic peptides from C. trachomatis, which aredisclosed in US Patent Application: 2014/0154287 and WO 2009/020553.Additionally, Chlamydia pneumoniae antigens including homologues of theforegoing polypeptides (see U.S. Pat. No. 6,919,187), can be used asantigens in the immunogenic compositions and methods as disclosedherein.

In some embodiments, an SA or non-SA antigen for use in the SA-MAPScomposition can be an intact (i.e., an entire or whole) antigen, or afunctional portion of an antigen that comprises more than one epitope.In some embodiments, an antigen is a peptide functional portion of anantigen. By “intact” in this context is meant that the antigen is thefull length antigen as that antigen polypeptide occurs in nature. Thisis in direct contrast to delivery of only a small portion or peptide ofthe antigen. Delivering an intact antigen to a cell enables orfacilitates eliciting an immune response to a full range of epitopes ofthe intact antigen, rather than just a single or selected few peptideepitopes. Accordingly, the methods and immunogenic compositionsdescribed herein encompass intact antigens associated with the polymerfor a more sensitive and have higher specificity of immune response ascompared to use of a single epitope peptide-based antigen.

Alternatively, in some embodiments, an intact SA antigen can be dividedinto many parts, depending on the size of the initial antigen.Typically, where a whole antigen is a multimer polypeptide, the wholeprotein can be divided into sub-units and/or domains where eachindividual sub-unit or domain of the antigen can be associated with thepolymer according to the methods as disclosed herein. Alternatively, insome embodiments, an intact SA antigen can be divided into functionalfragments, or parts, of the whole antigen, for example, at least two, orat least 3, or at least 4, or at least 5, or at least 6, or at least 7,or at least 8, or at least 9, or at least 10, or at least 11, or atleast 12, or at least 13, or at least 15, or at least 20, or at least25, or more than 25 portions (e.g., pieces or fragments), inclusive, andwhere each individual functional fragment of the antigen can beassociated with the polymer according to the methods as disclosedherein.

The fragmentation or division of a full length SA antigen polypeptidecan be an equal division of the full length antigen polypeptide, oralternatively, in some embodiments, the fragmentation is asymmetrical orunequal. As a non-limiting example, where an antigen is divided into twooverlapping fragments, an antigen can be divided into fragments ofapproximately the same (equal) size, or alternatively one fragment canbe about 45% of the whole antigen and the other fragment can be about65%. As further non-limiting examples, a whole antigen can be dividedinto a combination of differently sized fragments, for example, where anantigen is divided into two fragments, fragments can be divided intoabout 40% and about 70%, or about 45% and about 65%; or about 35% andabout 75%; or about 25% and about 85%, inclusive, of the whole antigen.Any combination of overlapping fragments of a full length whole antigenis encompassed for use in the generation of a panel of overlappingpolypeptides of an antigen. As an illustrative example only, where anantigen is divided into 5 portions, the portions can divided equally(i.e., each overlapping fragment is about 21% to 25% of the entire fulllength if the antigen) or unequally (i.e., an antigen can be dividedinto the following five overlapping fragments; fragment 1 is about 25%,fragment 2 is about 5%, fragment 3 is about 35%, fragment 4 is about 10%and fragment 5 is about 25% of the size of the full length antigen,provided each fragment overlaps with at least one other fragment).

Typically, a panel of antigen portions can substantially cover theentire length of the whole (or intact) antigen polypeptide. Accordingly,in some embodiments, an immunogenic composition comprises a polymer withmany different, and/or overlapping fragments of the same intact antigen.Overlapping protein fragments of an antigen can be produced much quickerand cheaper, and with increased stability as compared to the use ofpeptide antigens alone. Further in some embodiments, antigens which arepolypeptides larger than simple peptides are preferred as conformationis important for epitope recognition, and the larger antigenpolypeptides or fragments will provide a benefit over peptide fragments.

One of ordinary skill in the art can divide a whole antigen intooverlapping proteins of an antigen to create a panel of polypeptides ofthe antigen. By way of an illustrative example only, a SA antigen ClfAcan be divided into, for example at least 10 portions to generate apanel of 10 different polypeptides, each comprising a different butoverlapping ClfA-specific antigens fragments.

A target antigen for use in the methods and compositions describedherein can be expressed by recombinant means, and can optionally includean affinity or epitope tag to facilitate purification, which methods arewell-known in the art. Chemical synthesis of an oligopeptide, eitherfree or conjugated to carrier proteins, can be used to obtain antigen ofthe invention. Oligopeptides are considered a type of polypeptide. Anantigen can be expressed as a fusion with a complementary affinitymolecule, e.g., but not limited to rhizavidin or a derivative orfunctional fragment thereof. Alternatively, it is also possible toprepare target antigen and then conjugate it to a complementary affinitymolecule, e.g., but not limited to rhizavidin or a derivative orfunctional fragment thereof.

Polypeptides can also by synthesized as branched structures such asthose disclosed in U.S. Pat. Nos. 5,229,490 and 5,390,111. Antigenicpolypeptides include, for example, synthetic or recombinant B-cell andT-cell epitopes, universal T-cell epitopes, and mixed T-cell epitopesfrom one organism or disease and B-cell epitopes from another.

An antigen can be obtained through recombinant means or chemicalpolypeptide synthesis, as well as antigen obtained from natural sourcesor extracts, can be purified by means of the antigen's physical andchemical characteristics, such as by fractionation or chromatography.These techniques are well-known in the art.

In some embodiments, an antigen can be solubilized in water, a solventsuch as methanol, or a buffer. Suitable buffers include, but are notlimited to, phosphate buffered saline Ca²⁺/Mg²⁺ free (PBS), normalsaline (150 mM NaCl in water), and Tris buffer. Antigen not soluble inneutral buffer can be solubilized in 10 mM acetic acid and then dilutedto the desired volume with a neutral buffer such as PBS. In the case ofantigen soluble only at acid pH, acetate-PBS at acid pH can be used as adiluent after solubilization in dilute acetic acid. Glycerol can be asuitable non-aqueous solvent for use the compositions, methods and kitsdescribed herein.

Typically, when designing a protein vaccine against a pathogen, anextracellular protein or one exposed to the environment on a virus isoften the ideal candidate as the antigen component in the vaccine.Antibodies generated against that extracellular protein become the firstline of defense against the pathogen during infection. The antibodiesbind to the protein on the pathogen to facilitate antibody opsonizationand mark the pathogen for ingestion and destruction by a phagocyte suchas a macrophage. Antibody opsonization can also kill the pathogen byantibody-dependent cellular cytotoxicity. The antibody triggers arelease of lysis products from cells such as monocytes, neutrophils,eosinophils, and natural killer cells.

In one embodiment of the invention described herein, antigens for use inthe compositions as disclosed herein all wild type proteins, as in theamino acid residues have the sequences found in naturally occurringviruses and have not been altered by selective growth conditions ormolecular biological methods.

In one embodiment, the immunogenic compositions described as herein cancomprise antigens which are glycosylated proteins. In other words, anantigen of interest can each be a glycosylated protein. In oneembodiment of the immunogenic compositions as described herein,antigens, or antigen-fusion polypeptides are O-linked glycosylated. Inanother embodiment of the immunogenic compositions as described herein,antigens, or antigen-fusion polypeptides are N-linked glycosylated. Inyet another embodiment of the immunogenic compositions as describedherein, antigens, or antigen-fusion are both O-linked and N-linkedglycosylated. In other embodiments, other types of glycosylations arepossible, e.g., C-mannosylation. Glycosylation of proteins occurspredominantly in eukaryotic cells. N-glycosylation is important for thefolding of some eukaryotic proteins, providing a co-translational andpost-translational modification mechanism that modulates the structureand function of membrane and secreted proteins. Glycosylation is theenzymatic process that links saccharides to produce glycans, andattaches them to proteins and lipids. In N-glycosylation, glycans areattached to the amide nitrogen of asparagine side chain during proteintranslation. The three major saccharides forming glycans are glucose,mannose, and N-acetylglucosamine molecules. The N-glycosylationconsensus is Asn-Xaa-Ser/Thr, where Xaa can be any of the known aminoacids. O-linked glycosylation occurs at a later stage during proteinprocessing, probably in the Golgi apparatus. In O-linked glycosylation,N-acetyl-galactosamine, 0-fucose, 0-glucose, and/or N-acetylglucosamineis added to serine or threonine residues. One skilled in the art can usebioinformatics software such as NetNGlyc 1.0 and NetOGlyc Predictionsoftwares from the Technical University of Denmark to find the N- andO-glycosylation sites in a polypeptide in the present invention. TheNetNglyc server predicts N-Glycosylation sites in proteins usingartificial neural networks that examine the sequence context ofAsn-Xaa-Ser/Thr sequons. The NetNGlyc 1.0 and NetOGlyc 3.1 Predictionsoftware can be accessed at the EXPASY website. In one embodiment,N-glycosylation occurs in the target antigen polypeptide of the fusionpolypeptide described herein.

SA-Antigen-Fusion Proteins

In some embodiments, the SA antigen for use in the MAPS complex asdisclosed herein is fused to a recombinant biotin-binding protein. Insome embodiment, the recombinant biotin-binding protein is a rhizavidinprotein. In some embodiments, the Rhizavidin (Rhavi) protein comprisesSEQ ID NO: 1 or a protein or polypeptide of at least 85% amino acidsequence identity to SEQ ID NO: 1.

In some embodiments, the recombinant biotin-binding protein comprises anE. coli signal sequence fused to the N-terminus of an amino acidsequence comprising amino acids 45-179 of wild-type Rhizavidin (rhavi)which is as follows:

(SEQ ID NO: 1) FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD.

In some embodiments, the recombinant biotin-binding protein consists of,or consists essentially of, the amino acid sequence corresponding toamino acids 45-179 of the wild-type Rhizavidin. Amino acid sequence ofthe wild-type Rhizavidin is:

(SEQ ID NO: 21) MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALAFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD. In other words, the biotin-binding domain doesnot comprise (i.e., lacks) lacks amino acids 1-44 SEQ ID NO: 22)(MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALA,.

In some embodiments, the recombinant biotin-binding protein useful in afusion protein with at least one SA-antigen as disclosed hereincomprises an amino acid sequence having at least 50% identity, at least55% identity, at least 60% identity, at least 65% identity, at least 70%identity, at least 75% identity, at least 80% identity, preferably atleast 85% identity, at least 90% identity, at least 95% identity, atleast 96% identity, at least 97% identity, at least 98% identity, or atleast 99% identity, and more preferably at least 99.3% identity to SEQID NO: 1.

A SA-antigen for use in the SA-MAPS composition as disclosed herein canbe genetically fused to rhizavidin (rhavi), which is a dimericbiotin-binding protein from Rhizobium etli, according to the methods asdisclosed in U.S. Pat. No. 9,499,593 which is incorporated herein in itsentirety by reference.

In some embodiments, a biotin-binding protein useful in the SA-MAPScomposition as disclosed herein comprises a sequence X¹-X²-X³, whereinX² is a peptide having the amino acid sequence corresponding to aminoacids 45-179 of the wild-type Rhizavidin (i.e., SEQ ID NO: 1) and X¹ andX³ are independently absent, or a peptide of 1 to about 100 amino acidswith the proviso that the N-terminus of X¹ does not comprise an aminoacid sequence corresponding to N-terminus of amino acids 1-44 of thewild-type Rhizavidin.

In some embodiments, the biotin-binding proteins can comprise a signalpeptide conjugated to the N-terminus of the biotin-binding protein, i.e.X¹ can comprise a signal peptide. The signal peptide is also called aleader peptide in the N-terminus, which may or may not be cleaved offafter the translocation through the membrane. In some embodiments, theE. coli signal sequence is the Dsba signal sequence which comprises atleast MKKIWLALAGLVLAFSASA (SEQ ID NO: 23) or MKKIWLALAGLVLAFSASAAQDP(SEQ ID NO: 24). In some embodiments, the signal sequence isMKKVAAFVALSLLMAGC (SEQ ID NO: 25). Secretion/signal peptides aredescribed in more detail below. In some embodiments, the signal sequenceis MKKIWLALAGLVLAFSASA (SEQ ID NO: 26), MAPFEPLASGILLLLWLIAPSRA (SEQ IDNO: 27), MKKVAAFVALSLLMAGC (SEQ ID NO: 28), or a derivative orfunctional portion thereof. The signal sequence can be fused with thesequence comprising amino acids 45-179 of wild-type rhavi by a flexiblepeptide linker.

In some embodiments, the biotin-binding protein is a fusion protein withone or more SA-antigens. For example, the C-terminal of SEQ ID NO: 1 (ora protein of at least 80% or 85% or more sequence identity thereto) isfused to at least 1, or at least 2 or at least 3, or at least 4 or moreSA-antigens.

In some embodiments, a biotin-binding protein is a fusion proteincomprising a C-terminal of SEQ ID NO: 1 (or a protein of at least 80% or85% or more sequence identity thereto) is fused to any of hemolysin (HI)(e.g., hemolysin α or Hla209), Clumping factor A (ClfA), Clumping factorB (ClfB), serine-aspirate repeat protein D (SdrD), Iron regulatorsurface protein A (IsdA) and Iron regulator surface protein B (IsdB), orfragments thereof. In some embodiments, a biotin-binding protein is afusion protein comprising a the C-terminal of SEQ ID NO: 1 (or a proteinof at least 80% or 85% or more sequence identity thereto) fused to anyone of: Hla209(27-319), ClfA (221-559), ClfB (203-542), SdrD (246-682),IsdA (47-324), IsdB (48-447) or proteins or peptides having at least 85%sequence identity thereto.

Aspects of the present invention are directed to an isolated recombinantrhizavidin fusion protein comprising SEQ ID NO: 1 (or a protein of atleast 80% or 85% or more sequence identity thereto) fused toHla209(27-319) (Rhavi-Hla209). Aspects of the present invention aredirected to an isolated recombinant rhizavidin fusion protein comprisingSEQ ID NO: 1 (or a protein of at least 80% or 85% or more sequenceidentity thereto) fused to ClfA (221-559) (Rhavi-ClfA). Aspects of thepresent invention are directed to an isolated recombinant rhizavidinfusion protein comprising SEQ ID NO: 1 (or a protein of at least 80% or85% or more sequence identity thereto) fused to ClfB (203-542)(Rhavi-ClfB). Aspects of the present invention are directed to anisolated recombinant rhizavidin fusion protein comprising SEQ ID NO: 1(or a protein of at least 80% or 85% or more sequence identity thereto)fused to SdrD (246-682) (Rhavi-SdrD). Aspects of the present inventionare directed to an isolated recombinant rhizavidin fusion proteincomprising SEQ ID NO: 1 (or a protein of at least 80% or 85% or moresequence identity thereto) fused to IsdA (47-324) (Rhavi-IsdA). Aspectsof the present invention are directed to an isolated recombinantrhizavidin fusion protein comprising SEQ ID NO: 1 (or a protein of atleast 80% or 85% or more sequence identity thereto) fused to IsdB(48-447) (Rhavi-IsdB).

In some embodiments, a biotin-binding protein is a fusion proteincomprising a the C-terminal of SEQ ID NO: 1 (or a protein of at least80% or 85% or more sequence identity thereto) fused to at least twoantigens selected from any one of: Hla209(27-319), ClfA (221-559), ClfB(203-542), SdrD (246-682), IsdA (47-324), IsdB (48-447) or proteins orpeptides having at least 85% sequence identity thereto. The SA-antigensmay be the same antigens (e.g., SEQ ID NO: 1-A-A), or alternativelydifferent SA antigens (e.g., SEQ ID NO: 1-A-B), where A and B aredifferent SA-antigens. Exemplary Rhizavidin fusion proteins comprising 2SA-antigens are shown in Table 4.

TABLE 4 Exemplary Rhizavidin fusion proteins comprising differentcombinations of 2 SA-antigens. It is noted that the order of the 2antigens fused to the Rhizavidin protein of SEQ ID NO: 1 (referred to as“Rhavi”) or a homologue of at least 80% identity thereto can be in anyorder, e.g., Rhavi- HLA209-ClfA, or alternatively, Rhavi-ClfA-HLA209, orHla209-Rhavi-ClfA or ClfA-Rhavi-HLA209, for example. Rhavi-HLA209-CLFARhiva-HLA209-CLFB Rhiva-HLA209-SDRD Rhavi-HLA209-ISDA Rhavi-HLA209-ISDBRhavi-CLFA-CLFB Rhavi-CLFA-SDRD Rhavi-CLFA-ISDA Rhavi-CLFA-ISDBRhavi-CLFB-SDRD Rhavi-CLFB-ISDA Rhavi-CLFB-ISDB Rhavi-SDRD-ISDARhavi-SDRD-ISDB Rhavi-ISDA-ISDB

CLFA=CLFA protein or a fragment thereof, e.g., ClfA (221-559), CLFB=ClfBprotein or a fragment thereof, e.g., ClfB (203-542), SDRD=SdrD proteinor a fragment thereof, e.g., SdrD (246-682), ISDA=IsdA protein or afragment thereof, e.g., IsdA (47-324); ISDB=IsdB protein or a fragmentthereof, e.g., IsdB (48-477); HLA209=Hla protein with the 209 mutation,or a fragment thereof, e.g., Hla209(27-319). It is envisioned that anyof the SA antigens in the Rhavi-antigen-antigen fusion proteins shown inTable 4 can be substituted or replaced with any other SA antigen asdisclosed herein, or known to one of ordinary skill in the art.

In some embodiments, a rhizavidin fusion protein comprising a SA antigencan comprise a lipidation sequence at the N-terminus, e.g.,MKKVAAFVALSLLMAGC (SEQ ID NO: 29) or an amino acid 85% identity thereto.

In some embodiments, a rhizavidin fusion protein comprising a SA antigencan comprise a signal peptide linked to the N-terminus of thebiotin-binding domain either directly (e.g., via a bond) or indirectly(e.g., by a linker). In some embodiments, the signal peptide can belinked to the N-terminus of the biotin-binding domain by a peptidelinker. The peptide linker sequence can be of any length. For example,the peptide linker sequence can be one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen ormore amino acids in length. In some embodiments, the peptide linker isfour amino acids in length.

The peptide linker sequence can comprise any amino acid sequence. Forexample, the peptide linker can comprise an amino acid sequence whichcan be cleaved by a signal peptidase. In some embodiments, the peptidelinker comprises the amino acid sequence AQDP (SEQ ID NO: 30) or VSDP(SEQ ID NO: 31).

In some embodiments, a rhizavidin fusion protein comprising a SA antigencan be conjugated at its C-terminus to a peptide of 1-100 amino acids.Such peptides at the C-terminus can be used for purification tags,linkers to other domains, and the like. In some embodiments, arhizavidin fusion protein comprising a SA antigen comprises on its N- orC-terminus one or more (e.g., one, two, three, four, five, six, seven,eight, nine, ten or more) purification tags. Examples of purificationtags include, but are not limited to a histidine tag, a c-my tag, a Halotag, a Flag tag, and the like. In some embodiments, the biotin-bindingprotein comprises on its C-terminus a histidine tag, e.g. a (His)₆ (SEQID NO. 32). In some embodiments, a rhizavidin fusion protein comprisinga SA antigen for use in the SA-MAPS immunogenic composition as disclosedherein comprises a peptide of amino acid sequence GGGGSSSVDKLAAALEHHHHHH(SEQ ID NO: 33). This peptide at the C-terminus provides a histidine tagfor purification and a place for insertion of other domains, e.g.antigenic domains, in the biotin protein. Further, while Helppolainen etal. (Biochem J., 2007, 405: 397-405) describe expression of Rhizavidinin E. coli, there is no teaching or suggestion in Helppolainen et al.for conjugating an additional peptide to the C-terminus of thebiotin-binding domain of Rhizavidin.

A purification tag can be conjugated to a rhizavidin fusion proteincomprising a SA antigen as disclosed herein by a peptide linker toenhance the probability that the tag is exposed to the outside. Thelength of the linker can be at least one (e.g., one, two, three, four,five six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,or fifteen) amino acid. The linker peptide can comprise any amino acidsequence without limitations. In some embodiments, the linker peptidecomprises the amino acid sequence VDKLAAALE (SEQ ID NO: 34) orGGGGSSSVDKLAAALE (SEQ ID NO: 35). In some embodiments, a rhizavidinfusion protein comprising a SA antigen as disclosed herein can compriseat its C-terminus the amino acid sequence VDKLAAALEHHHHH (SEQ ID NO: 36)or GGGGSSSVDKLAAALEHHHHHH (SEQ ID NO: 37).

As discussed herein, a rhizavidin fusion protein comprising a SA antigenfor use in the SA-MAPS immunogenic composition as disclosed hereinconsists of amino acids 45-179 of wild-type Rhizavidin.

In some embodiments, rhizavidin fusion protein comprising a SA antigenfor use in the SA-MAPS immunogenic composition as disclosed herein cancomprise an N-terminal signal sequence as disclosed herein. In someembodiments, the signal sequence is attached to the N-terminal of thecomplementary affinity molecule as disclosed herein.

In some embodiments, a rhizavidin fusion protein comprising a SA antigenfor use in the SA-MAPS immunogenic composition as disclosed herein has aspacer peptide, e.g., a 14-residue spacer (GSPGISGGGGGILE) (SEQ ID NO:38) separating the SA antigen from the rhizavidin protein. The codingsequence of such a short spacer can be constructed by annealing acomplementary pair of primers. One of skill in the art can design andsynthesize oligonucleotides that will code for the selected spacer.Spacer peptides should generally have non-polar amino acid residues,such as glycine and proline.

Lipidated Rhizavidin Fusion Protein or Biotin-Binding Protein

In another aspect provided herein is a lipidated biotin-binding protein,e.g., a lipidated rhizavidin fusion protein comprising a SA antigen foruse in the SA-MAPS immunogenic composition as disclosed herein. As usedherein, the term “lipidated biotin-binding protein” refers to abiotin-binding protein that is covalently conjugated with a lipid. Thelipid moieties could be a diacyl or triacyl lipid.

In some embodiments, a rhizavidin fusion protein comprising a SA antigenfor use in the SA-MAPS immunogenic composition as disclosed hereincomprises a lipidation sequence. As used herein, the term “lipidationsequence” refers to an amino acid sequence that facilitates lipidationin bacteria, e.g., E. coli, of a polypeptide carrying the lipidatingsequence. The lipidation sequence can be present at the N-terminus orthe C-terminus of the protein. The lipidation sequence can be linked tothe recombinant biotin-binding protein to form a fusion protein, whichis in lipidated form when expressed in E. coli by conventionalrecombinant technology. In some embodiments, a lipidation sequence islocated at the N-terminus of the biotin-binding protein.

Any lipidation sequence known to one of ordinary skill in the art can beused. In some embodiments, the lipidating sequence is MKKVAAFVALSLLMAGC(SEQ ID NO: 39) or a derivative or functional portion thereof. Otherexemplary lididationg sequences include, but are not limited to,MNSKKLCCICVLFSLLAGCAS (SEQ ID NO: 40), MRYSKLTMLIPCALLLSAC (SEQ ID NO:41), MFVTSKKMTAAVLAITLAMSLSAC (SEQ ID NO: 42), MIKRVLVVSMVGLSLVGC (SEQID NO: 43), and derivatives or functional portions thereof.

In some embodiments, the lipidation sequence can be fused to arhizavidin fusion protein comprising a SA antigen via a peptide linker,wherein the peptide linker attaches the lipidating sequence to thebiotin-binding protein. In some embodiment, the peptide linker comprisesthe amino acid sequence VSDP (SEQ ID NO: 44) or AQDP (SEQ ID NO: 45).

In some embodiments, a rhizavidin fusion protein comprising a SA antigenfor use in the SA-MAPS immunogenic composition as disclosed herein thatis a lipoprotein as described herein have enhanced immunogenicity.Without wishing to be bound by a theory, lipid moieties at theN-terminals of the lipoproteins or lipopeptides contribute to theadjuvant activity. Accordingly, additional embodiments provideimmunogenic or vaccine compositions for inducing an immunologicalresponse, comprising the isolated biotin-binding lipoprotein, or asuitable vector for in vivo expression thereof, or both, and a suitablecarrier, as well as to methods for eliciting an immunological orprotective response comprising administering to a host the isolatedrecombinant biotin-binding lipoprotein, the vector expressing therecombinant biotin-binding lipoprotein, or a composition containing therecombinant lipoprotein or vector, in an amount sufficient to elicit theresponse.

A SA-MAPS immunogenic composition comprising a rhizavidin fusion proteincomprising a SA antigen that is a lipoprotein elicits an immunologicalresponse-local or systemic. The response can, but need not, beprotective.

Affinity Molecule Pairs

As disclosed herein, a key aspect of the SA-MAPS composition is theattachment of the SA antigens to the immunogenic polysaccharide. Asdiscussed herein, a SA antigen is connected to an immunogenicpolysaccharide via a complementary affinity pair. This connecting of theSA antigen to the immunogenic polysaccharide is mediated by theimmunogenic polysaccharide being connected to a first affinity molecule,which associates a second (e.g., complementary) affinity molecule, whichis attached to the SA antigen. An example complementary affinity pair isbiotin and a biotin-binding protein, e.g. biotin and rhizavidin proteinor fragment thereof.

Exemplary examples of the affinity complementary affinity pairs for usein the SA-MAPS immunogenic composition include, but without limitation,biotin binding proteins or avidin-like proteins that bind to biotin. Forexample, where the first affinity binding molecule is biotin (whichassociates with the polymer), the complementary affinity molecule can bea biotin binding protein or an avidin-like protein or a derivativethereof, e.g., but not limited to, avidin, rhizavidin, or streptavidinor variants, derivatives or functional portions thereof.

In some embodiments, the first affinity binding molecule is biotin, abiotin derivative, or a biotin mimic, for example, but not limited to,amine-PEG3-biotin (((+)-biotinylation-3-6,9-trixaundecanediamine) or aderivative or functional fragment thereof. A specific biotin mimetic hasa specific peptide motif containing sequence of DX_(a)AX_(b)PX_(c) (SEQID NO: 46), or CDX_(a)AX_(b)PX_(c)CG (SEQ ID NO: 47), where X_(a) is Ror L, X_(b) is S or T, and X_(c) is Y or W. These motifs can bind avidinand Neutravidin, but streptavidin. See. e.g., Gaj et al., 56 Prot.Express. Purif. 54 (2006). In some embodiments the first affinitybinding molecule is lipoic acid or a derivative thereof, or HABA(hydroxyazobenzene-benzoic acid, or dimethyl-HABA).

The linkage of the first affinity molecule to the immunogenicpolysaccharide, and the complementary affinity molecule to the SAantigen can be a non-covalent linkage, or a chemical mechanism, forinstance covalent binding, affinity binding, intercalation, coordinatebinding and complexation. Covalent binding provides for very stablebinding, and is particularly well-suited for the present embodiments.Covalent binding can be achieved either by direct condensation ofexisting side chains or by the incorporation of external bridgingmolecules.

For example, in some embodiments, a SA antigen can be non-covalentlybonded to one of the pairs in a complementary affixing pair. Inalternative embodiments, an antigen can be covalently bonded or fused toone of the pairs in a complementary affixing pair. Methods forgeneration of fusion proteins are well known in the art, and arediscussed herein.

In other embodiments, a first affinity binding molecule is linked to theimmunogenic polysaccharide by a non-covalent bond, or by a covalentbond. In some embodiments, a cross-linking reagent is used to covalentlybond the first affinity binding molecule to the immunogenicpolysaccharide as disclosed herein.

In some embodiments, the first affinity binding molecule associates withthe complementary affinity molecule by non-covalent bond association asknown in the art, including, but not limited to, electrostaticinteraction, hydrogen bound, hydrophobic interaction (i.e., van derWaals forces), hydrophilic interactions, and other non-covalentinteractions. Other higher order interactions with intermediate moietiesare also contemplated.

In some embodiments, the complementary affinity molecule is anavidin-related polypeptide. In specific embodiments, the complementaryaffinity molecule is rhizavidin, such as recombinant rhizavidin of SEQID NO: 1 or a protein having an amino acid that has at least 85%sequence identity to SEQ ID NO:1. In particular, the recombinantrhizavidin is a modified rhizavidin that can be expressed in E. coliwith a high yield. The typical yield is >30 mg per liter of E. coliculture. Rhizavidin has a lower sequence homology to egg avidin (22.4%sequence identity and 35.0% similarity) compared with other avidin-likeproteins. Use of the modified rhizavidin reduces the risk of the MAPSinducing an egg-related allergic reaction in a subject. Moreover,antibody to recombinant modified rhizavidin has no apparentcross-reactivity to egg avidin (and vice versa).

Additional affinity pairs that may be useful in the methods andcompositions described herein include antigen-antibody,metal/ion-metal/ion-binding protein, lipid/lipid binding protein,saccharide/saccharide binding protein, amino acid/peptide/amino acid orpeptide binding protein, enzyme-substrate or enzyme-inhibitor,ligand-agonist/receptor, or biotin mimetic. When using alternativeaffinity pairs, alternative means of attaching the respective polymerand antigen may also be employed, such as in vitro enzymatic reactionsrather than genetic fusion. More specifically, antigen-antibody affinitypair provides for a very strong and specific interaction. The antigencan be any epitope including protein, peptide, nucleic acid, lipid,poly/oligosaccharide, ion, etc. The antibody can be any type ofimmunoglobulin, or the Ag-binding portion of an immunoglobulin, such asa Fab fragment. Regarding metal/ion-metal/ion binding protein, examplesinclude Ni NTA vs. histidine-tagged protein, or Zn vs. Zn bindingprotein. Regarding lipid/lipid binding protein, examples includecholesterol vs. cholesterol binding protein. Regardingsaccharide/saccharide binding protein, examples include maltose vs.maltose binding protein, mannose/glucose/oligosaccharide vs. lectin.Enzyme-substrate/inhibitors include substrates from a wide range ofsubstances, including protein, peptide, amino acid, lipid, sugar, orions. The inhibitor can be the analog of the real substrate which cangenerally bind to the enzymes more tightly and even irreversibly. Forexample, trypsin vs. soy trypsin inhibitor. The inhibitor can be naturalor synthetic molecule. Regarding other ligand/agonist-receptor, ligandcan be from a wide range of substance, including protein, peptide, aminoacid, lipid, sugar, ion, agonist can be the analog of the real ligand.Examples include the LPS vs. TLR4 interaction.

Cross-Linking Reagents

Many bivalent or polyvalent linking agents are useful in coupling atleast one or more affinity molecules to the immunogenic polysaccharideof the SA-MAPS immunogenic composition as disclosed herein. For example,representative coupling agents can include organic compounds such asthioesters, carbodiimides, succinimide esters, disocyanates,glutaraldehydes, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. See Killen & Lindstrom, 133 J. Immunol. 1335 (1984);Jansen et al., 62 Imm. Rev. 185 (1982); Vitetta et al.

In some embodiments, cross-linking reagents agents described in theliterature are encompassed for use in the methods, immunogeniccompositions and kits as disclosed herein. See. e.g., Ramakrishnan, etal., 44 Cancer Res. 201 (1984) (describing the use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester)); Umemoto et al., U.S.Pat. No. 5,030,719 (describing the use of a halogenated acetyl hydrazidederivative coupled to an antibody by way of an oligopeptide linker).Particular linkers include: (a) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (b) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (c) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat#21651G); (d) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (f) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkages or linking agents described above contain components thathave different attributes, thus leading to conjugates with differingphysio-chemical properties. For example, sulfo-NHS esters of alkylcarboxylates are more stable than sulfo-NHS esters of aromaticcarboxylates. NHS-ester containing linkers are less soluble thansulfo-NHS esters. Further, the linker SMPT contains a stericallyhindered disulfide bond, and can form conjugates with increasedstability. Disulfide linkages, are in general, less stable than otherlinkages because the disulfide linkage can be cleaved in vitro,resulting in less conjugate available. Sulfo-NHS, in particular, canenhance the stability of carbodimide couplings. Carbodimide couplings(such as EDC) when used in conjunction with sulfo-NHS, forms esters thatare more resistant to hydrolysis than the carbodimide coupling reactionalone.

Additional cross linkers for —SH (thiolated CP) to —NH₂ linkages includebut are not limited to: sulfa-LC-SMPT; sulfo-LC-SMPT(4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate));sulfo-KMUS (N-[k-maleimidoundecanoyloxy]sulfosuccinimide ester);sulfo-LC-SPDP (sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate) which cleaves by thiols;sulfo-SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate); sulfo-SIAB(N-sulfosuccinimidyl[4-iodoacetyl]aminobenzoate); sulfa-EMCS([N-e-maleimidocaproyloxy]sulfosuccinimide ester); EMCA(N-e-maleimidocaproic acid); sulfo-SMCC (sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate); sulfo-MBS(m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester); sulfo-GMBS(N-[g-maleimidobutyryloxy]sulfosuccinimide ester); BMPA(N-.beta.-maleimidopropionic acid); 2-immunothiolane hydrochloride;3-(2-pyridyldithio)propionic acid N-succinimidyl ester;3-malemidopropionic acid N-succinimidyl ester; 4-maleimidobutyric acidN-succinimidyl ester; SMPT(4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene); LC-SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate-]);KMUA (N-k-maleimidoundecanoic acid); LC-SPDP (succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate); SMPH(succinimidyl-6-[.beta.-maleimidopropionamido]hexanoate); SMPB(succinimidyl 4-[p-maleimidophenyl]butyrate); SIAB(N-succinimidyl[4-iodoacetyl]aminobenzoate); EMCS([N-e-Maleimidocaproyloxy]succinimide ester); SMCC (succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate); MBS(i-Maleimidobenzoyl-N-hydroxysuccinimide ester); SBAP (succinimidyl3-[bromoacetamido]propionate); BMPS(N-[.beta.-maleimidopropyloxylsuccinimide ester); AMASN-(a-maleimidoacetoxy)succinimide ester); SIA (N-succinimidyliodoacetate); and N-succinimidyl (4-iodoacetyl)-aminobenzoate.

The agents can also be crosslinked using crosslinkers for —SH to —OHgroups. Such cross linkers include but are not limited to PMPI(N-[p-maleimidophenyl]isocyanate).

Exemplary cross-linking molecules for use in the methods and immunogeniccompositions as disclosed herein include, but are not limited to thoselisted in Tables 5 and 6.

TABLE 5 Exemplary homobifunctional crosslinkers* Crosslinker ReactiveGroups, Crosslinking Target Features Example Products Amine-to-Amine NHSesters DSG; DSS; BS3; TSAT (trifunctional); Bioconjugate Toolkit ReagentPairs NHS esters, PEG spacer BS(PEG)5; BS(PEG)9 NHS esters,thiol-cleavable DSP; DTSSP NHS esters, misc-cleavable DST; BSOCOES; EGS;Sulfo-EGS Imidoesters DMA; DMP; DMS Imidoesters, thiol-cleavable DTBPOther DFDNB; THPP (trifunctional); Aldehyde-Activated Dextran KitSulfhydryl-to-Sulfhydryl Maleimides BMOE; BMB; BMH; TMEA (trifunctional)Maleimides, PEG spacer BM(PEG)2; BM(PEG)3 Maleimides, cleavable BMDB;DTME Pyridyldithiols (cleavable) DPDPB Other HBVS (vinylsulfone)Nonselective Aryl azides BASED (thiol-cleavable) *crosslinking reagentsthat have the same type of reactive group at either end. Reagents areclassified by what chemical groups they cross link (left column) andtheir chemical composition (middle column). Products are listed in orderof increasing length within each cell.

TABLE 6 Exemplary heterobifunctional crosslinkers* Crosslinker ReactiveCrosslinking Targets Groups, Features Example ProductsAmine-to-Sulfhydryl NHS ester/Maleimide AMAS; BMPS; GMBS and Sulfo-GMBS; MBS and Sulfo-MBS; SMCC and Sulfo-SMCC; EMCS and Sulfo-EMCS; SMPBand Sulfo- SMPB; SMPH; LC-SMCC; Sulfo- KMUS NHS ester/Maleimide,SM(PEG)2; SM(PEG)4; SM(PEG)6; PEG spacer SM(PEG)8; SM(PEG)12; SM(PEG)24NHS ester/Pyridyldithiol, SPDP; LC-SPDP and Sulfo-LC- CrosslinkingTargets Crosslinker Reactive Example Products Groups, Features cleavableSPDP; SMPT; Sulfo-LC-SMPT NHS esters/Haloacetyl SIA; SBAP; SIAB;Sulfo-SIAB Amine-to-Nonselective NHS ester/Aryl Azide NHS-ASA ANB-NOSSulfo-HSAB Sulfo-NHS-LC-ASA SANPAH and Sulfo-SANPAH NHS ester/ArylAzide, Sulfo-SFAD; Sulfo-SAND; Sulfo- cleavable SAED NHS ester/DiazirineSDA and Sulfo-SDA; LC-SDA and Sulfo-LC-SDA NHS ester/Diazirine, SDAD andSulfo-SDAD cleavable Amine-to-Carboxyl Carbodiimide DCC; EDCSulfhydryl-to-Nonselective Pyridyldithiol/Aryl Azide APDPSulfhydryl-to-Carbohydrate Maleimide/Hydrazide BMPH; EMCH; MPBH; KMUHPyridyldithiol/Hydrazide BMPH; EMCH; MPBH; KMUHCarbohydrate-to-Nonselective Hydrazide/Aryl Azide ABHHydroxyl-to-Sulfhydryl Isocyanate/Maleimide PMPI Amine-to-DNA NHSester/Psoralen SPB *crosslinking reagents that have the differentreactive groups at either end. Reagents are classified by what chemicalgroups they cross ink (left column) and their chemical composition(middle column), Products are listed in order of increasing lengthwithin each cell.

Co-Stimulatory Factor

In some embodiments, an immunogenic composition comprising the SA-MAPSas disclosed herein comprises at least one co-stimulatory molecule. Insome embodiments, the co-stimulatory factor is cross-linked to theimmunogenic polysaccharide. In some embodiments, the co-stimulatoryfactor is associated to the immunogenic polysaccharide by acomplementary affinity pair similar to how the SA antigen is associatedwith the immunogenic polysaccharide. In some embodiments, where thecomplementary affinity pair which links the co-stimulatory factor to theimmunogenic polysaccharide is the same, or a different complementaryaffinity pair which links the SA antigen to the immunogenicpolysaccharide.

In some embodiments, at least one, or at least 2, or at least 3, or atleast 5, or at least 10, or at least 15, or at least 20, or at least 50,or at least 100, or more than about 100, inclusive, co-stimulatoryfactors can be associated with the immunogenic polysaccharide asdisclosed herein. In some embodiments, the co-stimulatory factors can bethe same co-stimulator factor, or they can be a variety of differentco-stimulatory factors associated with the immunogenic polysaccharide.

In some embodiments, the co-stimulator factor is a ligand/agonist ofToll like receptors, e.g., but not limited to TLR1, TLR2, TLR3, TLR4,TLRS, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, etc. In some embodiments, aco-stimulator factor is a NOD ligand/agonist, or an activator/agonist ofthe inflammasome. Without wishing to be bound by theory, theinflammasome is a multiprotein oligomer consisting of caspase 1, PYCARD,NALP and sometimes caspase 5 or caspase 11 and promotes the maturationof inflammatory cytokines interleukin 1-03 and interleukin 18.

In some embodiments, a co-stimulator factor is a cytokine. In someembodiments, a cytokine is selected from the group consisting of:GM-CSF; IL-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10;IL-12; IL-23; IFN-α; IFN-β; IFN-γ; MIP-1α; MIP-1β; TGF-β; TNFα, andTNFβ. In some embodiments, the co-stimulatory factor is an adjuvant,which may be associated with the polymer, as just discussed, or may beadded to the MAPS composition prior to or concurrent with administrationto a subject. Adjuvants are further described elsewhere herein.

Production of SA Antigens and SA Antigens Fused to the ComplementaryAffinity Molecule

Recombinant proteins may be conveniently expressed and purified by aperson skilled in the art, or by using commercially available kits, forexample PROBOND™ Purification System (Invitrogen Corp., Carlsbad,Calif.). In some embodiments, recombinant antigens can be synthesizedand purified by protein purification methods using bacterial expressionsystems, yeast expression systems, baculovirus/insect cell expressionsystem, mammalian cell expression systems, or transgenic plant or animalsystems as known to persons of ordinary skill in the art.

The fusion polypeptides as described herein, e.g., a SA antigen fused toa rhizavidin protein of SEQ ID NO: 1 (e.g., Rhavi-Hla209(27-319),Rhavi-ClfA (221-559), Rhavi-ClfB (203-542), Rhavi-SdrD (246-682),Rhavi-IsdA (47-324), Rhavi-IsdB (48-447)) can all be synthesized andpurified by protein and molecular methods that are well known to oneskilled in the art. Molecular biology methods and recombinantheterologous protein expression systems are used. For example,recombinant protein can be expressed in bacteria, mammalian, insect,yeast, or plant cells; or in transgenic plant or animal hosts.

In one embodiment, provided herein is an isolated polynucleotideencoding a fusion polypeptide or a non-fusion polypeptide describedherein. Conventional polymerase chain reaction (PCR) cloning techniquescan be used to construct a chimeric or fusion coding sequence encoding afusion polypeptide as described herein. A coding sequence can be clonedinto a general purpose cloning vector such as pUC19, pBR322,pBLUESCRIPT® vectors (Stratagene, Inc.) or pCR TOPO® (Invitrogen). Theresultant recombinant vector carrying the nucleic acid encoding apolypeptide as described herein can then be used for further molecularbiological manipulations such as site-directed mutagenesis to create avariant fusion polypeptide as described herein or can be subcloned intoprotein expression vectors or viral vectors for protein synthesis in avariety of protein expression systems using host cells selected from thegroup consisting of mammalian cell lines, insect cell lines, yeast,bacteria, and plant cells.

Each PCR primer should have at least 15 nucleotides overlapping with itscorresponding templates at the region to be amplified. The polymeraseused in the PCR amplification should have high fidelity such asPfuULTRA® polymerase (Stratagene) for reducing sequence mistakes duringthe PCR amplification process. For ease of ligating several separate PCRfragments together, for example in the construction of a fusionpolypeptide, and subsequently inserting into a cloning vector, the PCRprimers should also have distinct and unique restriction digestion siteson their flanking ends that do not anneal to the DNA template during PCRamplification. The choice of the restriction digestion sites for eachpair of specific primers should be such that the fusion polypeptidecoding DNA sequence is in-frame and will encode the fusion polypeptidefrom beginning to end with no stop codons. At the same time the chosenrestriction digestion sites should not be found within the coding DNAsequence for the fusion polypeptide. The coding DNA sequence for theintended polypeptide can be ligated into cloning vector pBR322 or one ofits derivatives, for amplification, verification of fidelity andauthenticity of the chimeric coding sequence, substitutions/or specificsite-directed mutagenesis for specific amino acid mutations andsubstitutions in the polypeptide.

Alternatively, the coding DNA sequence for the polypeptide can be PCRcloned into a vector using for example, the TOPO® cloning methodcomprising topoisomerase-assisted TA vectors such as pCR®-TOPO,pCR®-Blunt II-TOPO, pENTR/D-TOPO®, and pENTR/SD/D-TOPO® (Invitrogen,Inc., Carlsbad, Calif.). Both pENTR/D-TOPO®, and pENTR/SD/D-TOPO® aredirectional TOPO entry vectors which allow the cloning of the DNAsequence in the 5′-3′ orientation into a GATEWAY® expression vector.Directional cloning in the 5′-3′ orientation facilitates theunidirectional insertion of the DNA sequence into a protein expressionvector such that the promoter is upstream of the 5′ ATG start codon ofthe fusion polypeptide coding DNA sequence, enabling promoter drivenprotein expression. The recombinant vector carrying the coding DNAsequence for the fusion polypeptide can be transfected into andpropagated in general cloning E. coli such as XL1Blue, SURE®(STRATAGENE®) and TOP-10 cells (Invitrogen).

One skilled in the art would be able to clone and ligate the codingregion of the antigen of interest with the coding region of thecomplementary affinity molecule to construct a chimeric coding sequencefor a fusion polypeptide comprising the antigen or a fragment thereofand the complementary affinity molecule of a derivative thereof usingspecially designed oligonucleotide probes and polymerase chain reaction(PCR) methodologies that are well known in the art. One skilled in theart would also be able to clone and ligate the chimeric coding sequencefor a fusion protein into a selected vector, e.g., bacterial expressionvector, an insect expression vector or baculovirus expression vector.The coding sequences of antigen and the target antigen polypeptide orfragment thereof should be ligated in-frame and the chimeric codingsequence should be ligated downstream of the promoter, and between thepromoter and the transcription terminator. Subsequent to that, therecombinant vector is transfected into regular cloning E. coli, such asXL1Blue. Recombinant E. coli harboring the transfer vector DNA is thenselected by antibiotic resistance to remove any E. coli harboringnon-recombinant plasmid DNA. The selected transformant E. coli are grownand the recombinant vector DNA can be subsequently purified fortransfection into S. frugiperda cells.

In some embodiments, the SA antigens as disclosed herein can comprise asignal peptide for translocation into periplasmic space of bacteria. Thesignal peptide is also called a leader peptide in the N-terminus, whichmay or may not be cleaved off after the translocation through themembrane. One example of a signal peptide is MKKIWLALAGLVLAFSASA (SEQ IDNO: 23) as disclosed herein. Another signal sequence isMAPFEPLASGILLLLWLIAPSRA (SEQ ID NO: 48). Other examples of signalpeptides can be found at SPdb, a Signal Peptide Database, which is foundat the world wide web site of “proline.bic.nus.edu.sg/spdb/”.

In some embodiments, where the antigen is fused to a complementaryaffinity protein, the signal sequence can be located at the N-terminalof the complementary affinity protein. For example, if an antigen isfused to an avidin-like protein, the signal sequence can be located atthe N-terminal of the complementary affinity protein. In someembodiments, the signal sequence is cleaved off from the complementaryaffinity protein before the complementary affinity protein associateswith the first affinity molecule.

In some embodiments, a SA antigen and/or complementary affinity proteinas described herein lacks a signal sequence.

The polypeptides described herein can be expressed in a variety ofexpression host cells e.g., bacteria, yeasts, mammalian cells, insectcells, plant cells, algal cells such as Chlamadomonas, or in cell-freeexpression systems. In some embodiments the nucleic acid can besubcloned from the cloning vector into a recombinant expression vectorthat is appropriate for the expression of fusion polypeptide inbacteria, mammalian, insect, yeast, or plant cells or a cell-freeexpression system such as a rabbit reticulocyte expression system. Somevectors are designed to transfer coding nucleic acid for expression inmammalian cells, insect cells and year in one single recombinationreaction. For example, some of the GATEWAY® (Invitrogen) destinationvectors are designed for the construction of baculovirus, adenovirus,adeno-associated virus (AAV), retrovirus, and lentiviruses, which uponinfecting their respective host cells, permit heterologous expression offusion polypeptides in the appropriate host cells. Transferring a geneinto a destination vector is accomplished in just two steps according tomanufacturer's instructions. There are GATEWAY® expression vectors forprotein expression in insect cells, mammalian cells, and yeast.Following transformation and selection in E. coli, the expression vectoris ready to be used for expression in the appropriate host.

Examples of other expression vectors and host cells are the strong CMVpromoter-based pcDNA3.1 (Invitrogen) and pCINEO vectors (Promega) forexpression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat,and MCF-7; replication incompetent adenoviral vector vectors pADENO-X™,pAd5F35, pLP-ADENO™-X-CMV (CLONTECH®), pAd/CMVN5-DEST, pAd-DEST vector(Invitrogen) for adenovirus-mediated gene transfer and expression inmammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for usewith the RETRO-XT™ system from Clontech for retroviral-mediated genetransfer and expression in mammalian cells; pLenti4N5-DEST™,pLenti6N5-DEST™, and pLenti6.2N5-GW/lacZ (Invitrogen) forlentivirus-mediated gene transfer and expression in mammalian cells;adenovirus-associated virus expression vectors such as pAAV-MCS,pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associatedvirus-mediated gene transfer and expression in mammalian cells; BACpak6baculovirus (Clontech) and pFASTBAC™ HT (Invitrogen) for the expressionin S. frugiperda 9 (Sf9), Sf11, Tn-368 and BTI-TN-5B4-1 insect celllines; pMT/BiPN5-His (Invitrogen) for the expression in Drosophilaschneider S2 cells; Pichia expression vectors pPICZα, pPICZ, pFLDα andpFLD (Invitrogen) for expression in P. pastoris and vectors pMETα andpMET for expression in P. methanolica; pYES2/GS and pYD1 (Invitrogen)vectors for expression in yeast S. cerevisiae.

Recent advances in the large scale expression heterologous proteins inChlamydomonas reinhardtii are described. Griesbeck., 34 Mol. Biotechnol.213 (2006); Fuhrmann, 94 Methods Mol Med. 191 (2006). Foreignheterologous coding sequences are inserted into the genome of thenucleus, chloroplast and mitochondria by homologous recombination. Thechloroplast expression vector p64 carrying the most versatilechloroplast selectable marker aminoglycoside adenyl transferase (aadA),which confer resistance to spectinomycin or streptomycin, can be used toexpress foreign protein in the chloroplast. The biolistic gene gunmethod can be used to introduce the vector in the algae. Upon its entryinto chloroplasts, the foreign DNA is released from the gene gunparticles and integrates into the chloroplast genome through homologousrecombination.

Also included in the invention are complementary affinity molecule fusedto an antigen. In some embodiments, the fusion construct can alsooptionally comprise purification tags, and/or secretion signal peptides.These fusion proteins may be produced by any standard method. Forexample, for production of a stable cell line expressing anantigen-complementary affinity molecule fusion protein, PCR-amplifiedantigen nucleic acids may be cloned into the restriction site of aderivative of a mammalian expression vector. For example, KA, which is aderivative of pcDNA3 (Invitrogen) contains a DNA fragment encoding aninfluenza virus hemagglutinin tag (HA). Alternatively, vectorderivatives encoding other tags, such as c-myc or poly Histidine tags,can be used. The antigen-complementary affinity molecule fusionexpression construct may be co-transfected, with a marker plasmid, intoan appropriate mammalian cell line (e.g., COS, HEK293T, or NIH 3T3cells) using, for example, LIPOFECTAMINE™ (Gibco-BRL, Gaithersburg, Md.)according to the manufacturer's instructions, or any other suitabletransfection technique known in the art. Suitable transfection markersinclude, for example, β-galactosidase or green fluorescent protein (GFP)expression plasmids or any plasmid that does not contain the samedetectable marker as the antigen-complementary affinity molecule fusionprotein. The fusion protein expressing cells can be sorted and furthercultured, or the tagged antigen-complementary affinity molecule fusionprotein can be purified. In some embodiments, an antigen-complementaryaffinity molecule fusion protein is amplified with a signal peptide. Inalternative embodiments, a cDNA encoding an antigen-complementaryaffinity molecule fusion protein can be amplified without the signalpeptide and subcloned into a vector (pSecTagHis) having a strongsecretion signal peptide. In another example, antigen-complementaryaffinity molecule fusion protein can have an alkaline phosphatase (AP)tag, or a histadine (His) tag for purification. Any method known topersons of ordinary skill in the art for protein purification of theantigen and/or antigen-complementary affinity molecule fusion protein isencompassed for use in the methods of the invention.

In some embodiments, any of the polypeptides described herein isproduced by expression from a recombinant baculovirus vector. In anotherembodiment, any of the polypeptides described herein is expressed by aninsect cell. In yet another embodiment, any of the polypeptidesdescribed herein is isolated from an insect cell. There are severalbenefits of protein expression with baculovirus in insect cells,including high expression levels, ease of scale-up, production ofproteins with posttranslational modifications, and simplified cellgrowth. Insect cells do not require CO₂ for growth and can be readilyadapted to high-density suspension culture for large-scale expression.Many of the post-translational modification pathways present inmammalian systems are also utilized in insect cells, allowing theproduction of recombinant protein that is antigenically,immunogenically, and functionally similar to the native mammalianprotein.

Baculoviruses are DNA viruses in the family Baculoviridae. These virusesare known to have a narrow host-range that is limited primarily toLepidopteran species of insects (butterflies and moths). The baculovirusAutographa californica Nuclear Polyhedrosis Virus (AcNPV), which hasbecome the prototype baculovirus, replicates efficiently in susceptiblecultured insect cells. AcNPV has a double-stranded closed circular DNAgenome of about 130,000 base-pairs and is well characterized with regardto host range, molecular biology, and genetics. The BaculovirusExpression Vector System (BEVS) is a safe and rapid method for theabundant production of recombinant proteins in insect cells and insects.Baculovirus expression systems are powerful and versatile systems forhigh-level, recombinant protein expression in insect cells. Expressionlevels up to 500 mg/l have been reported using the baculovirusexpression system, making it an ideal system for high-level expression.Recombinant baculoviruses that express foreign genes are constructed byway of homologous recombination between baculovirus DNA and chimericplasmids containing the gene sequence of interest. Recombinant virusescan be detected by virtue of their distinct plaque morphology andplaque-purified to homogeneity.

Recombinant fusion proteins described herein can be produced in insectcells including, but not limited to, cells derived from the Lepidopteranspecies S. frugiperda. Other insect cells that can be infected bybaculovirus, such as those from the species Bombyx mori, Galleriamellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as asuitable substrate to produce recombinant proteins described herein.Baculovirus expression of recombinant proteins is well known in the art.See U.S. Pat. Nos. 4,745,051; 4,879,236; 5,179,007; 5,516,657;5,571,709; 5,759,809. It will be understood by those skilled in the artthat the expression system is not limited to a baculovirus expressionsystem. What is important is that the expression system directs theN-glycosylation of expressed recombinant proteins. The recombinantproteins described herein can also be expressed in other expressionsystems such as Entomopox viruses (the poxviruses of insects),cytoplasmic polyhedrosis viruses (CPV), and transformation of insectcells with the recombinant gene or genes constitutive expression. A goodnumber of baculovirus transfer vectors and the correspondingappropriately modified host cells are commercially available, forexample, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 fromBD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from NOVAGEN®,and pPolh-FLAG and pPolh-MAT from SIGMA ALDRICH®.

The region between the promoter and the transcriptional terminator canhave multiple restriction enzyme digestion sites for facilitatingcloning of the foreign coding sequence, in this instance, the coding DNAsequence for an antigen polypeptide, and a complementary affinitymolecule. Additional sequences can be included, e.g., signal peptidesand/or tag coding sequences, such as His-tag, MAT-Tag, FLAG tag,recognition sequence for enterokinase, honeybee melittin secretionsignal, beta-galactosidase, glutathione S-transferase (GST) tag upstreamof the MCS for facilitating the secretion, identification, properinsertion, positive selection of recombinant virus, and/or purificationof the recombinant protein.

Standard techniques known to those of skill in the art can be used tointroduce mutations (to create amino acid substitutions in an antigenpolypeptide sequence of the fusion polypeptide described herein, e. g.,in the antigen in the nucleotide sequence encoding the fusionpolypeptide described herein, including, for example, site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, the variant fusionpolypeptide has less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions, inclusive, relative to the fusion polypeptidesdescribed herein.

Certain silent or neutral missense mutations can also be made in the DNAcoding sequence that do not change the encoded amino acid sequence orthe capability to promote transmembrane delivery. These types ofmutations are useful to optimize codon usage, or to improve recombinantprotein expression and production.

Specific site-directed mutagenesis of a coding sequence for the fusionpolypeptide in a vector can be used to create specific amino acidmutations and substitutions. Site-directed mutagenesis can be carriedout using, e. g., the QUICKCHANGE® site-directed mutagenesis kit fromStratagene according to the manufacturer's instructions.

In one embodiment, described herein are expression vectors comprisingthe coding DNA sequence for the polypeptides described herein for theexpression and purification of the recombinant polypeptide produced froma protein expression system using host cells selected from, e.g.,bacteria, mammalian, insect, yeast, or plant cells. The expressionvector should have the necessary 5′ upstream and 3′ downstreamregulatory elements such as promoter sequences, ribosome recognition andTATA box, and 3′ UTR AAUAAA transcription termination sequence forefficient gene transcription and translation in its respective hostcell. The expression vector is, preferably, a vector having thetranscription promoter selected from a group consisting of CMV(cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, β-actinpromoter, SV40 (simian virus 40) promoter and muscle creatine kinasepromoter, and the transcription terminator selected from a groupconsisting of SV40 poly(A) and BGH terminator; more preferably, anexpression vector having the early promoter/enhancer sequence ofcytomegalovirus and the adenovirus tripartite leader/intron sequence andcontaining the replication origin and poly(A) sequence of SV40. Theexpression vector can have additional coding regions, such as thoseencoding, for example, 6X-histidine, V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (Honeybeemelittin,□ α-factor, PHO, Bip), which can be incorporated into theexpressed fusion polypeptide. In addition, there can be enzyme digestionsites incorporated after these coding regions to facilitate theirenzymatic removal if they are not needed. These additional nucleic acidsare useful for the detection of fusion polypeptide expression, forprotein purification by affinity chromatography, enhanced solubility ofthe recombinant protein in the host cytoplasm, and/or for secreting theexpressed fusion polypeptide out into the culture media or thespheroplast of the yeast cells. The expression of the fusion polypeptidecan be constitutive in the host cells or it can be induced, e.g., withcopper sulfate, sugars such as galactose, methanol, methylamine,thiamine, tetracycline, infection with baculovirus, and(isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analogof lactose.

In another embodiment, the expression vector comprising a polynucleotidedescribed herein is a viral vector, such as adenovirus, adeno-associatedvirus (AAV), retrovirus, and lentivirus vectors, among others.Recombinant viruses provide a versatile system for gene expressionstudies and therapeutic applications.

In some embodiments, the fusion polypeptides described herein areexpressed from viral infection of mammalian cells. The viral vectors canbe, for example, adenovirus, adeno-associated virus (AAV), retrovirus,and lentivirus. A simplified system for generating recombinantadenoviruses is presented by He et al., 95 PNAS 2509 (1998). The gene ofinterest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. Theresultant plasmid is linearized by digesting with restrictionendonuclease PmeI, and subsequently cotransformed into E. coli. BJ5183cells with an adenoviral backbone plasmid, e.g. pADEASY-1 ofStratagene's ADEASY™ Adenoviral Vector System. Recombinant adenovirusvectors are selected for kanamycin resistance, and recombinationconfirmed by restriction endonuclease analyses. Finally, the linearizedrecombinant plasmid is transfected into adenovirus packaging cell lines,for example HEK 293 cells (E1-transformed human embryonic kidney cells)or 911 (E1-transformed human embryonic retinal cells). Fallaux, et al. 7Human Gene Ther. 215 (1996). Recombinant adenovirus are generated withinthe HEK 293 cells.

Recombinant lentivirus has the advantage of delivery and expression offusion polypeptides in dividing and non-dividing mammalian cells. TheHIV-1 based lentivirus can effectively transduce a broader host rangethan the Moloney Leukemia Virus (MoMLV)-based retroviral systems.Preparation of the recombinant lentivirus can be achieved using, forexample, the pLenti4N5-DEST™, pLenti6N5-DEST™ or pLenti vectors togetherwith VIRAPOWER™ Lentiviral Expression systems from Invitrogen, Inc.

Recombinant adeno-associated virus (rAAV) vectors are applicable to awide range of host cells including many different human and non-humancell lines or tissues. rAAVs are capable of transducing a broad range ofcell types and transduction is not dependent on active host celldivision. High titers, >10⁸ viral particle/ml, are easily obtained inthe supernatant and 10¹¹-10¹² viral particle/ml with furtherconcentration. The transgene is integrated into the host genome soexpression is long term and stable.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying the codingnucleic acid, AAV RC vector containing AAV rep and cap genes, andadenovirus helper plasmid pDF6, into 50×150 mm plates of subconfluent293 cells. Cells are harvested three days after transfection, andviruses are released by three freeze-thaw cycles or by sonication.

AAV vectors can be purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin. Auricchio et. al., 12 Human Gene Ther. 71 (2001); Summerford &Samulski, 72 J. Virol. 1438 (1998); Summerford & Samulski, 5 Nat. Med.587 (1999). AAV2/1 and AAV2/5 vectors are currently purified by threesequential CsCl gradients.

Without wishing to be bound to theory, when proteins are expressed by acell, including a bacterial cell, the proteins are targeted to aparticular part in the cell or secreted from the cell. Thus, proteintargeting or protein sorting is the mechanism by which a cell transportsproteins to the appropriate positions in the cell or outside of it.Sorting targets can be the inner space of an organelle, any of severalinterior membranes, the cell's outer membrane, or its exterior viasecretion. This delivery process is carried out based on informationcontained in the protein itself. Correct sorting is crucial for thecell; errors can lead to diseases.

With some exceptions, bacteria lack membrane-bound organelles as foundin eukaryotes, but they may assemble proteins onto various types ofinclusions such as gas vesicles and storage granules. Also, depending onthe species of bacteria, bacteria may have a single plasma membrane(Gram-positive bacteria), or both an inner (plasma) membrane and anouter cell wall membrane, with an aqueous space between the two calledthe periplasm (Gram-negative bacteria). Proteins can be secreted intothe environment, according to whether or not there is an outer membrane.The basic mechanism at the plasma membrane is similar to the eukaryoticone. In addition, bacteria may target proteins into or across the outermembrane. Systems for secreting proteins across the bacterial outermembrane may be quite complex and play key roles in pathogenesis. Thesesystems may be described as type I secretion, type II secretion, etc.

In most Gram-positive bacteria, certain proteins are targeted for exportacross the plasma membrane and subsequent covalent attachment to thebacterial cell wall. A specialized enzyme, sortase, cleaves the targetprotein at a characteristic recognition site near the proteinC-terminus, such as an LPXTG motif (SEQ ID NO: 19) (where X can be anyamino acid), then transfers the protein onto the cell wall. A systemanalogous to sortase/LPXTG, having the motif PEP-CTERM (SEQ ID NO: 49),termed exosortase/PEP-CTERM, is proposed to exist in a broad range ofGram-negative bacteria.

Proteins with appropriate N-terminal targeting signals are synthesizedin the cytoplasm and then directed to a specific protein transportpathway. During, or shortly after its translocation across thecytoplasmic membrane, the protein is processed and folded into itsactive form. Then the translocated protein is either retained at theperiplasmic side of the cell or released into the environment. Since thesignal peptides that target proteins to the membrane are keydeterminants for transport pathway specificity, these signal peptidesare classified according to the transport pathway to which they directproteins. Signal peptide classification is based on the type of signalpeptidase (SPase) that is responsible for the removal of the signalpeptide. The majority of exported proteins are exported from thecytoplasm via the general “Secretory (Sec) pathway”. Most well knownvirulence factors (e.g. exotoxins of Staphylococcus aureus, protectiveantigen of Bacillus anthracis, lysteriolysin O of Listeriamonocytogenes) that are secreted by Gram-positive pathogens have atypical N-terminal signal peptide that would lead them to theSec-pathway. Proteins that are secreted via this pathway aretranslocated across the cytoplasmic membrane in an unfolded state.Subsequent processing and folding of these proteins takes place in thecell wall environment on the trans-side of the membrane. In addition tothe Sec system, some Gram-positive bacteria also contain the Tat-systemthat is able to translocate folded proteins across the membrane.Pathogenic bacteria may contain certain special purpose export systemsthat are specifically involved in the transport of only a few proteins.For example, several gene clusters have been identified in mycobacteriathat encode proteins that are secreted into the environment via specificpathways (ESAT-6) and are important for mycobacterial pathogenesis.Specific ATP-binding cassette (ABC) transporters direct the export andprocessing of small antibacterial peptides called bacteriocins. Genesfor endolysins that are responsible for the onset of bacterial lysis areoften located near genes that encode for holin-like proteins, suggestingthat these holins are responsible for endolysin export to the cell wall.Wooldridge, BACT. SECRETED PROTS: SECRETORY MECHS. & ROLE IN PATHOGEN.(Caister Academic Press, 2009)

In some embodiments, the signal sequence useful in the present inventionis OmpA Signal sequence, however any signal sequence commonly known bypersons of ordinary skill in the art which allows the transport andsecretion of antimicrobial agents outside the bacteriophage infectedcell are encompassed for use in the present invention.

Signal sequence that direct secretion of proteins from bacterial cellsare well known in the art, for example as disclosed in Internationalapplication WO 2005/071088. For example, one can use some of thenon-limited examples of signal peptide shown in Table 7, which can beattached to the amino-terminus or carboxyl terminus of the antimicrobialpeptide (Amp) or antimicrobial polypeptide to be expressed by theantimicrobial-agent engineered bacteriophage, e.g., AMP-engineeredbacteriophage. Attachment can be via fusion or chimera composition withselected antigen or antigen-complementary affinity molecule fusionprotein resulting in the secretion from the bacterium infected with theantimicrobial-agent engineered bacteriophage, e.g. AMP-engineeredbacteriophage.

TABLE 7Example signal peptides to direct secretion of a protein or peptide antigen orantigen-complementary affinity molecule fusion protein of a bacterial cellSecretion Signal Peptide Amino Acid Pathway sequence (NH₂-CO₂) GeneGenus/Species secA1 MKKIMLVITLILVSPIAQQTEAK Hly (LLO)Listeria monocytogenes D (SEQ ID NO: 50) MKKKIISAILMSTVILSAAAPLSG Usp45Lactococcus lactis VYADT (SEQ ID NO: 51) MKKRKVLIPLMALSTILVSSTGNPag (protective Bacillus anthracis LEVIQAEV (SEQ ID NO: 52) antigen)secA2 MNMKKATIAATAGIAVTAFAAP Iap (invasion- Listeria monocytogenesTIASAST (SEQ ID NO: 53) associated protein p60) MQKTRKERILEALQEEKKNKKSNamA Imo2691 Listeria monocytogenes KKFKTGATIAGVTAIATSITVPGI (autolysin)EVIVSADE (SEQ ID NO: 54) MKKLKMASCALVAGLMFSGLT *BA_0281Bacillus anthracis PNAFAED (SEQ ID NO: 55) (NLP/P60 family)MAKKFNYKLPSMVALTLVGSA * atl (autolysin) Staphylococcus aureusVTAHQVQAAE (SEQ ID NO: 56) Tat MTDKKSENQTEKTETKENKGM Imo0367Listeria monocytogenes TRREMLKLSAVAGTGIAVGATG LGTILNVVDQVDKALT (SEQ IDNO: 57) MAYDSRFDEWVQKLKEESFQN PhoD (alkaline Bacillus subtillisNTFDRRKFIQGAGKIAGLGLGLT phosphatase) IAQSVGAFG (SEQ ID NO: 58)

The polypeptides as described herein, e.g., antigens orantigen-complementary affinity molecule fusion protein can be expressedand purified by a variety methods known to one skilled in the art, forexample, the fusion polypeptides described herein can be purified fromany suitable expression system. Fusion polypeptides can be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others; which arewell-known in the art. See. e.g., Scopes, PROTEIN PURIFICATION:PRINCIPLES & PRACTICE (1982); U.S. Pat. No. 4,673,641.

A number of procedures can be employed when recombinant proteins arepurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to the protein of choice. With theappropriate ligand, the protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,the protein of choice can be purified using affinity or immunoaffinitycolumns.

After the protein is expressed in the host cells, the host cells can belysed to liberate the expressed protein for purification. Methods oflysing the various host cells are featured in “Sample Preparation-Toolsfor Protein Research” EMD Bioscience and in the Current Protocols inProtein Sciences (CPPS). An example purification method is affinitychromatography such as metal-ion affinity chromatograph using nickel,cobalt, or zinc affinity resins for histidine-tagged fusionpolypeptides. Methods of purifying histidine-tagged recombinant proteinsare described by Clontech using their TALON® cobalt resin and byNOVAGEN® in their pET system manual, 10th edition. Another preferredpurification strategy is immuno-affinity chromatography, for example,anti-myc antibody conjugated resin can be used to affinity purifymyc-tagged fusion polypeptides. When appropriate protease recognitionsequences are present, fusion polypeptides can be cleaved from thehistidine or myc tag, releasing the fusion polypeptide from the affinityresin while the histidine-tags and myc-tags are left attached to theaffinity resin.

Standard protein separation techniques for purifying recombinant andnaturally occurring proteins are well known in the art, e.g., solubilityfractionation, size exclusion gel filtration, and various columnchromatography.

Solubility fractionation: Often as an initial step, particularly if theprotein mixture is complex, an initial salt fractionation can separatemany of the unwanted host cell proteins (or proteins derived from thecell culture media) from the protein of interest. The preferred salt isammonium sulfate. Ammonium sulfate precipitates proteins by effectivelyreducing the amount of water in the protein mixture. Proteins thenprecipitate on the basis of their solubility. The more hydrophobic aprotein is, the more likely it is to precipitate at lower ammoniumsulfate concentrations. A typical protocol includes adding saturatedammonium sulfate to a protein solution so that the resultant ammoniumsulfate concentration is between 20-30%. This concentration willprecipitate the most hydrophobic of proteins. The precipitate is thendiscarded (unless the protein of interest is hydrophobic) and ammoniumsulfate is added to the supernatant to a concentration known toprecipitate the protein of interest. The precipitate is then solubilizedin buffer and the excess salt removed if necessary, either throughdialysis or diafiltration. Other methods that rely on solubility ofproteins, such as cold ethanol precipitation, are well known to those ofskill in the art and can be used to fractionate complex proteinmixtures.

Size exclusion filtration: The molecular weight of the protein of choicecan be used to isolate it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,AMICON® or MILLIPORE® membranes). As a first step, the protein mixtureis ultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column chromatography: The protein of choice can also be separated fromother proteins on the basis of its size, net surface charge,hydrophobicity, and affinity for ligands. In addition, antibodies raisedagainst recombinant or naturally occurring proteins can be conjugated tocolumn matrices and the proteins immunopurified. All of these methodsare well known in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).For example, an antigen polypeptide can be purified using a PA63heptamer affinity column. Singh et al., 269, J. Biol. Chem. 29039(1994).

In some embodiments, a combination of purification steps comprising, forexample: (a) ion exchange chromatography, (b) hydroxyapatitechromatography, (c) hydrophobic interaction chromatography, and (d) sizeexclusion chromatography can be used to purify the fusion polypeptidesdescribed herein.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. Commercially available cell-free expressionsystems include the TNT coupled reticulocyte lysate Systems (Promega)which uses rabbit reticulocyte-based in vitro system.

Determining the Efficacy of a SA-MAPS Immunogenic Composition:

The effectiveness of a SA-MAPS immunogenic composition as disclosedherein can be measured either by proliferation assays, by cytolyticassays, such as chromium release assays to measure the ability of aT-cell to lyse its specific target cell, or by measuring the levels ofB-cell activity by measuring the levels of circulating antibodiesspecific for the antigen in serum. An immune response may also bedetected by measuring the serum levels of antigen specific antibodyinduced following administration of the antigen, and more specifically,by measuring the ability of the antibodies so induced to enhance theopsonophagocytic ability of particular white blood cells, as describedherein. The level of protection of the immune response may be measuredby challenging the immunized host with the antigen that has beenadministered. For example, if the antigen to which an immune response isdesired is a bacterium, the level of protection induced by theimmunogenic amount of the antigen is measured by detecting the percentsurvival or the percent mortality after challenge of the animals withthe bacterial cells. In one embodiment, the amount of protection may bemeasured by measuring at least one symptom associated with the bacterialinfection, e.g., a fever associated with the infection. The amount ofeach of the antigens in the multi-antigen or multi-component vaccine orimmunogenic compositions will vary with respect to each of the othercomponents and can be determined by methods known to the skilledartisan. Such methods would include procedures for measuringimmunogenicity and/or in vivo efficacy. In certain embodiments, the term“about” leans within 20%, preferably within 10%, and more preferablywithin 5%.

In some embodiments, the invention further provides antibodies andantibody compositions which bind specifically and selectively to theSA-MAPS immunogenic composition as disclosed herein. In someembodiments, antibodies are generated upon administration of a SA-MAPSimmunogenic composition as disclosed herein to a subject. In someembodiments, the antibodies of the present invention are functional asmeasured by killing bacteria in either an animal efficacy model or viaan opsonophagocytic killing assay. In some embodiments, the antibodiesof the invention confer passive immunity to a subject. The presentinvention further provides polynucleotide molecules encoding an antibodyor antibody fragment of the invention, and a cell, cell line (such ashybridoma cells or other engineered cell lines for recombinantproduction of antibodies) or a transgenic animal that produces anantibody or antibody composition of the invention, using techniqueswell-known to those of skill in the art.

Antibodies or antibody compositions of the invention may be used in amethod of treating or preventing a Staphylococcal infection, disease orcondition associated with a Staphylococcus sp. in a subject, the methodcomprising generating a polyclonal or monoclonal antibody preparation,and using said antibody or antibody composition to confer passiveimmunity to the subject. Antibodies of the invention may also be usefulfor diagnostic methods, e.g., detecting the presence of or quantifyingthe levels of CP5, CP8 or a conjugate thereof.

Several animal models known in the art may be used to assess theefficacy of any one of the SA-MAPS immunogenic composition as disclosedherein. For example:

Passive Murine Sepsis Model: Mice are passively immunizedintraperitoneally (i.p.) with SA-MAPS immunogenic composition asdisclosed herein. The mice are challenged 24 hours later with a lethaldose of S. aureus. The bacterial challenge is administered intravenously(i.v. or i.p.) ensuring that any survival could be attributed to thespecific in vivo interaction of the antibody with the bacteria. Thebacterial challenge dose is determined to be the dose required toachieve lethal sepsis of approximately 20% of the un-immunized controlmice. Statistical evaluation of survival studies can be carried out byKaplan-Meier analysis.

Active Immunization and Challenge Model: In this model, mice areactively immunized subcutaneously (s.c.) with a SA-MAPS immunogeniccomposition as disclosed herein at 0, 3 and 6 weeks (or a similarschedule known to those skilled in the art) and challenged with S.aureus at week 8 (or other similar schedule known to those skilled inthe art) by the intravenous or intraperitoneal route. The bacterialchallenge dose is calibrated to achieve approximately 20% survival inthe control group over a 14 day period. Statistical evaluation ofsurvival studies can be carried out by Kaplan-Meier analysis.

Passive Infectious Endocarditis Model: A passive immunization model forinfectious endocarditis (IE) caused by S. aureus has previously beenused to show that ClfA can induce protective immunity. See, Vemachio etal. (2006) Antmicro. Agents & Chemo. 50:511-518. In this model of IE,rabbits or rats are used to simulate clinical infections that include acentral venous catheter, bacteremia, and hematogenous seeding to distalorgans. Catheterized rabbits or rats with sterile aortic valvevegetations are administered a SA-MAPS immunogenic composition asdisclosed herein. After 24 hours, the animals are challenged i.v. withheterologous staphylococcal strains or a MRSA strain. Then 48 hoursafter challenge, cardiac vegetations, kidneys and blood are harvestedand cultured. The frequency of staphylococcal infection in cardiac valvevegetations, kidneys, and blood is then measured. In one study, whenanimals were challenged with either MRSE ATCC 35984 or MRSA PFESA0003,significant reductions in infection rate were shown using either thepolyclonal antibody preparation or the monoclonal antibody to ClfA. See,Vemachio et al., supra.

Passive Infectious Endocarditis Model: The infectious endocarditis modelhas also been adapted for active immunization studies. Rabbits or ratsare immunized intramuscularly (i.m.) with a SA-MAPS immunogeniccomposition as disclosed herein and challenged with aureus two weekslater via the i.v. route.

Pyelonephritis Model: In the pyelonephritis model, mice are immunized onweeks 0, 3 and 6 (or a similar schedule known to those skilled in theart) with a SA-MAPS immunogenic composition as disclosed herein. On week8, the animals are challenged by, e.g., i.p. injection of, e.g., 1.7×10⁸cfu S. aureus PFESA0266. After 48 hours, the kidneys and/or othertissues are harvested and cultured. Finally, colony forming units ofchallenge bacteria are enumerated in the kidneys and/or other tissues.This model evaluates systemic dissemination in the animal.

Monitoring Functional Antibodies Using Opsonophagocytic Killing Assays:Differentiated effector cells from a cell line (e.g. HL60s) orpolymorphonuclear cells (PMNs) isolated from donor human blood usingLYMPHOLYTE®-poly solation (Cedarlane laboratories limited, Ontario,Canada) as per manufacturer's protocol can be used for this assay.Effector cells were resuspended in assay buffer (Modified Eagle's mediacontaining 1% bovine serum albumin) at approximately 2×10⁷ cells/mlconcentration and placed in 37° C. incubator until ready to use. S.aureus strain PFESA0266 was grown overnight on tryptic soy agar plates.Bacterial cells were scraped, washed twice and resuspended in assaybuffer containing 5% glycerol to an OD⁶⁰⁰⁼¹, which equals toapproximately 5.times.10.sup.8 cfu/ml concentration. One ml aliquots ofthe bacterial suspension were frozen and stored at −40° C. until readyto use. Frozen bacterial suspension were thawed and adjusted to aconcentration of 10×10⁶ cfu/ml in assay buffer and placed on ice. Theassay was performed using a sterile 96 deep well 1 ml polypropyleneplates. Two fold serial dilutions of antibody samples (50 μl) wereprepared and followed by addition of 300 μl of assay buffer to theantibody mix. Bacteria were added (50 μl) to the plates and placed on arotary shaker at 4° C. for 30 minutes. The opsonization step wasfollowed by addition of 50 μl of human complement (1% finalconcentration). Finally, 50 μl of effector cells (10×10⁷ cells/mlconcentration) were added to the plate and the suspension mixed well byrepeated pipetting. A 50 μl aliquot of the suspension was 10 foldserially diluted in sterile 1% saponin solution, vortexed to minimizebacterial clumping and plated on tryptic soy agar in duplicate. Theassay plate was incubated at 37° C. for 1 hour with continuous mixingusing rotisserie style shaker. At the end of the incubation a 50 μlaliquot of suspension was 10 fold serially diluted in sterile 1% saponinsolution, mixed by vortexing to minimize bacterial clumping and platedon tryptic soy agar in duplicate. The percentage killing was calculatedby determining the ratio of the number of cfu surviving at 60 minutes inwells with bacteria, antibodies, complement and effector cells to thenumber of cfu surviving in tubes lacking antibodies but containingbacteria, complement and effector cells. Controls containing bacteria,complement, and sera were included to adjust for any reduction in cfudue to clumping.

Complement Adsorption: Serum from human donors adsorbed against S.aureus strains PFESA0266, PFESA0286 and PFESA0270 can be used as asource of complement in the assay. S. aureus strains were grownovernight on TSA plates at 37° C. Cells were scraped from the plate andresuspended in sterile PBS. Bacterial cells were centrifuged at 10,000rpm for 10 minutes at 4° C. and cell pellet was resuspended in humanserum for adsorption. Serum was incubated with bacteria on a nutator at4° C. for 30 minutes. Cells were centrifuged, serum transferred toanother tube containing bacteria and the adsorption step repeated againfor 30 minutes. Finally, the cells were centrifuged and the serum passedthrough a 0.2 micron filter before 0.5 ml aliquots were frozen down inliquid nitrogen.

Method II—OPA Using HL-60 Cells: HL-60 cells were differentiatedaccording to S. Romero-Steiner, et al., Clin Diagn Lab Immunol 4 (4)(1997), pp. 415-422. Harvested HL-60 cells were resuspended in assaybuffer (Modified Eagle's media containing 1% bovine serum albumin) atapproximately 10.sup.8 cells/nil and placed in 37° C. incubator untilready to use. S. aureus was grown overnight on tryptic soy agar plates.Bacterial cells were scraped, washed twice and resuspended in assaybuffer containing 5% glycerol to an OD⁶⁰⁰ ⁼¹, which equals toapproximately 5×10⁸ cfu/ml. One ml aliquots of the bacterial suspensionwere frozen and stored at −40° C. until ready to use. Frozen bacterialsuspension were thawed and adjusted to a concentration of 10×10⁶ cfu/mlin assay buffer and placed on ice. The assay was performed using asterile 96 deep well 1 ml polypropylene plates. Two fold serialdilutions of monoclonal antibody samples (25 μl) were prepared andfollowed by addition of 150 μl of assay buffer to the antibodysuspension. Bacteria were added (25 μl) to the plates and placed on arotary shaker at 4° C. for 30 minutes followed by addition of 25 μl ofhuman complement (1% final concentration). Finally, 25 μl of HL-60 cells(10×10⁷ cells/ml) were added to the plate and the suspension mixed wellby repeated pipetting. A 25 μl aliquot of the suspension was 10 foldserially diluted in sterile 1% saponin solution, mixed by vortexing tominimize bacterial clumping and plated on tryptic soy agar induplicates. The assay plate was incubated at 37° C. for 1 hour withcontinuous mixing using rotisserie style shaker. At the end ofincubation a 25 μl aliquot of suspension was 10 fold serially diluted insterile 1% saponin solution, mixed by vortexing to and plated on trypticsoy agar in duplicate. The percentage killing was calculated bydetermining the ratio of the number of cfu surviving at 60 minutes inwells with bacteria, antibodies, complement and HL-60 cells to thenumber of cfu surviving in tubes lacking antibodies but containingbacteria, complement and HL-60 cells. Controls containing bacteriacomplement and mAb was included to adjust for any reduction in cfu dueto clumping.

Formulations of an Immune Composition and Methods of Use

Specific embodiments of the present invention provide for use of theSA-MAPS immunogenic compositions as disclosed herein to elicit an immuneresponse to S. aureus in an animal. More specifically, the compositionselicit both humoral and cellular immunity, and in many instance mucosalimmunity. Embodiments of the present invention provide at least partialprotection from or partial improvement after infection by, inparticular, S. aureus.

In one embodiment, provided herein is a method of vaccinating a mammalcomprising administering the SA-MAPS immunogenic composition comprisingat least one, or multiple SA antigens attached to an immunogenicpolysaccharide for use in eliciting an immune response to the one ormore antigens attached to the polymer when administered to a subject. Insome embodiments, the immune response is a humoral and/or cellularimmune response.

Accordingly, one aspect of the present invention relates to methods toelicit an immune response in a subject, comprising administering to thesubject a SA-MAPS immunogenic composition comprising at least one typeof immunogenic polysaccharide (e.g., CP5, CP8, a CP5-CP8 conjugate,pneumococcal PS1 (CP 1) etc., at least one SA antigen, and at least onecomplementary affinity-molecule pair comprising (i) a first affinitymolecule which associates with the immunogenic polysaccharide, and (ii)a complementary affinity molecule which associates with the SA antigen,to attach the SA antigen to the immunogenic polysaccharide, (e.g., thefirst affinity molecule associates with the complementary affinitymolecule to link the SA antigen to the immunogenic polysaccharide).

Accordingly, one aspect of the present invention relates to methods toelicit a humoral and/or cellular immunity to multiple SA antigens at thesame time, e.g., where the immunogenic composition administered to thesubject comprises an immunogenic polysaccharide comprising at least 1,or at least 2, or a more, e.g., a plurality of the same or different SAantigens.

One aspect of the present invention relates to a method of immunizationor vaccinating a subject, e.g., a bird or a mammal, e.g., a humanagainst S. aureus comprising administering a SA-MAPS immune compositionas disclosed herein comprising at least one SA antigen derived from oneor more pathogens. In some embodiments, a subject can be immunizedagainst at least 1, or at least 2, or at least 2, or at least 3, or atleast 5, or at least 10, or at least 15, or at least about 20, or atleast 50, or at least about 100, or more than 100 different SA antigensat the same time, where the immunogenic polysaccharide of the SA-MAPSimmunogenic composition has different SA antigens attached.

In some embodiments, a subject can be administered several differentSA-MAPS immunogenic compositions as disclosed herein, for example, asubject can be administered a SA-MAPS composition comprising animmunogenic polysaccharide with a SA antigen, or a plurality of SAantigens, e.g., antigens A, B, C, and D etc., and also administered aSA-MAPS composition comprising an immunogenic polysaccharide comprisinga different SA antigen, or a different set of SA antigens, e.g.,antigens W, X, Y, and Z etc. Alternatively, a subject can beadministered a SA-MAPS composition comprising a immunogenicpolysaccharide A (e.g., CP5) with an SA antigen, or a plurality of SAantigens, e.g., antigens A, B, C, and D, etc., and also administered aSA-MAPS composition comprising a immunogenic polysaccharide B (e.g. CP8)comprising the same e.g., antigens A, B, C, and D etc., or a differentset of antigens. It is envisioned that the present invention provides amethod for the immunization of a subject with as many SA antigens asdesired, e.g., with a variety of different immunogenic complexes asdescribed herein, to enable immunization with as many as 100 or moreantigens.

In one embodiment, the SA-MAPS immunogenic compositions as describedherein comprise a pharmaceutically acceptable carrier. In anotherembodiment, the SA-MAPS immunogenic composition described herein isformulated for administering to a bird, mammal, or human, as or in avaccine. Suitable formulations can be found in, for example, Remington'sPharmaceutical Sciences (2006), or Introduction to Pharmaceutical DosageForms (4th ed., Lea & Febiger, Philadelphia, 1985).

In one embodiment, the SA-MAPS immunogenic compositions as describedherein comprise pharmaceutically acceptable carriers that are inherentlynontoxic and nontherapeutic. Examples of such carriers include ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol. For all administrations, conventional depot forms are suitablyused. Such forms include, for example, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, sublingual tablets,and sustained release preparations. For examples of sustained releasecompositions, see U.S. Pat. Nos. 3,773,919, 3,887,699, EP 58,481A, EP158,277A, Canadian Patent No. 1176565; Sidman et al., 22 Biopolymers 547(1983); Langer et al., 12 Chem. Tech. 98 (1982). The proteins willusually be formulated at a concentration of about 0.1 mg/ml to 100 mg/mlper application per patient.

In one embodiment, other ingredients can be added to vaccineformulations, including antioxidants, e.g., ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

In some embodiments, the SA-MAPS immunogen composition as disclosedherein is administered with at least one adjuvant or an immunemodulator, or both. Adjuvants are a heterogeneous group of substancesthat enhance the immunological response against an antigen that isadministered simultaneously. In some instances, adjuvants improve theimmune response so that less vaccine is needed. Adjuvants serve to bringthe antigen—the substance that stimulates the specific protective immuneresponse—into contact with the immune system and influence the type ofimmunity produced, as well as the quality of the immune response(magnitude or duration). Adjuvants can also decrease the toxicity ofcertain antigens; and provide solubility to some vaccine components.Almost all adjuvants used today for enhancement of the immune responseagainst antigens are particles or form particles together with theantigen. In the book VACCINE DESIGN—SUBUNIT & ADJUVANT APPROACH (Powell& Newman, Eds., Plenum Press, 1995), many known adjuvants are describedboth regarding their immunological activity and regarding their chemicalcharacteristics. The type of adjuvants that do not form particles are agroup of substances that act as immunological signal substances and thatunder normal conditions consist of the substances that are formed by theimmune system as a consequence of the immunological activation afteradministration of particulate adjuvant systems.

Adjuvants for immunogenic compositions and vaccines are well known inthe art. Examples include, but not limited to, monoglycerides and fattyacids (e. g. a mixture of mono-olein, oleic acid, and soybean oil);mineral salts, e.g., aluminium hydroxide and aluminium or calciumphosphate gels; oil emulsions and surfactant based formulations, e.g.,MF59 (microfluidised detergent stabilized oil-in-water emulsion), QS21(purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),MPL-SE, Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);particulate adjuvants, e.g., virosomes (unilamellar liposomal vehiclesincorporating influenza haemagglutinin), AS04 ([SBAS4] Al salt withMPL), ISCOMS (structured complex of saponins and lipids), polylactideco-glycolide (PLG); microbial derivatives (natural and synthetic), e.g.,monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton),AGP [RC-529] (synthetic acylated monosaccharide), Detox-PC, DC_Chol(lipoidal immunostimulators able to self-organize into liposomes),OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotidescontaining immunostimulatory CpG motifs), or other DNA structures,modified LT and CT (genetically modified bacterial toxins to providenon-toxic adjuvant effects); endogenous human immunomodulators, e.g.,hGM-CSF or hIL-12 (cytokines that can be administered either as proteinor plasmid encoded), Immudaptin (C3d tandem array), MoGM-CSF,TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59and inert vehicles, such as gold particles. Additional adjuvants areknown in the art, see, e.g., U.S. Pat. No. 6,890,540; U. S. Patent Pub.No. 2005/0244420; PCT/SE97/01003.

Additional suitable adjuvants used to enhance an immune response of theSA-MAPS composition as disclosed herein further include, withoutlimitation, MPL™ (3-O-deacylated monophosphoryi lipid A, Corixa;Hamilton, Mont.), which is described in U.S. Pat. No. 4,912,094. Alsosuitable for use as adjuvants are synthetic lipid A analogs oraminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and those that aredescribed in U.S. Pat. No. 6,113,918. One such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R1-3-t-etradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529). This 529 adjuvantis formulated as an aqueous form (AF) or as a stable emulsion (SE).Still other adjuvants include muramyl peptides, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-hydroxyphosphoryloxy)-ethylamine (MTP-PE);oil-in-water emulsions, such as MF59 (U.S. Pat. No. 6,299,884)(containing 5% Squalene, 0.5% polysorbate 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microlluidics, Newton, Mass.)), and SAF (containing 10%Squalene, 0.4% polysorbate 80, 5% pluronic-blocked polymer L121, andthr-MDP, either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion); incomplete Freund's adjuvant(IFA); aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate; Amphigen; Avridine; L121/squalene;D-lactide-polylactide/glycoside; pluronic polyols; killed Bordetella;saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, Mass.),described in U.S. Pat. No. 5,057,540, Iscomatrix® (CSL Limited,Parkville, Australia), described in U.S. Pat. No. 5,254,339, andimmunostimulating complexes (ISCOMS); Mycobacterium tuberculosis;bacterial lipopolysaccharides; synthetic polynucleotides such asoligonucleotides containing a CpG motif (e.g., U.S. Pat. No. 6,207,646);IC-31 (Intercell AG, Vienna, Austria), described in EP Patent Nos.1,296,713 and 1,326,634; a pertussis toxin (PT) or mutant thereof acholera toxin or mutant thereof (e.g., U.S. Pat. Nos. 7,285,281,7,332,174, 7,361,355 and 7,384,640); or an E. coli heat-labile toxin(LT) or mutant thereof, particularly LT-K63, LT-R72 (e.g., U.S. Pat.Nos. 6,149,919, 7,115,730 and 7,291,588).

In some embodiments, the SA-MAPS immunogen composition as disclosedherein is administered with at least one immune modulator. An“immunomodulator” or “immune modulator” is an agent that perturb oralter the immune system, such that either up-regulation ordown-regulation of humoral and/or cell-mediated immunity is observed. Inone embodiment, up-regulation of the humoral and/or cell-mediated armsof the immune system is provided. Examples of certain immunomodulatorsinclude, e.g., an adjuvant or cytokine, or Iscomatrix™ (CSL Limited;Parkville, Australia), described in U.S. Pat. No. 5,254,339 amongothers. Non-limiting examples of adjuvants that can be used in theimmunogenic composition of the present invention include the RIBIadjuvant system (Ribi Inc.; Hamilton, Mont.), alum, mineral gels such asaluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsionssuch as, e.g., Freund's complete and incomplete adjuvants, Blockcopolymer (CytRx; Atlanta, Ga.), QS-21 (Cambridge Biotech Inc.;Cambridge, Mass.), SAF-M (Chixon; Emeryville, Calif.), Amphigen™.adjuvant, saponin, Quil A or other saponin fraction, monophosphoryllipid A, and Avridine lipid-amine adjuvant. Non-limiting examples ofoil-in-water emulsions useful in the immunogenic composition of theinvention include modified SEAM62 and SEAM 1/2 formulations. ModifiedSEAM62 is oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1%(v/v) Span® 85 Detergent (ICI Surfactants), 0.7% (v/v) polysorbate 80detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 mcg/ml Quil A, 100mcg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is anoil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) Span® 85Detergent, 0.7% v/v) polysorbate 80 detergent, 2.5% (v/v) ethanol, 100mcg/ml Quil A, and 50 mcg/ml cholesterol. Other “immunomodulators” thatcan be included in the immunogenic composition include, e.g., one ormore interleukins, interferons, or other known cytokines or chemokines.In one embodiment, the adjuvant may be a cyclodextrin derivative or apolyanionic polymer, such as those described in U.S. Pat. Nos. 6,165,995and 6,610,310, respectively. It is to be understood that theimmunomodulator and/or adjuvant to be used will depend on the subject towhich the immunogenic composition will be administered, the route ofinjection and the number of injections to be given.

In some embodiments, the SA-MAPS immunogen composition as disclosedherein is administered with at least one immune modulator. A number ofcytokines or lymphokines have been shown to have immune modulatingactivity, and thus may be useful in a manner the same or similar toadjuvants, including, but not limited to, the interleukins 1-.alpha.,1-.beta., 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127),13, 14, 15, 16, 17 and 18 (and its mutant forms); the interferons-α, βand γ; granulocyte-macrophage colony stimulating factor (GM-CSF) (see,e.g., U.S. Pat. No. 5,078,996 and ATCC Accession Number 39900);macrophage colony stimulating factor (M-CSF); granulocyte colonystimulating factor (G-CSF); and the tumor necrosis factors α and β.Still other adjuvants that are useful with the immunogenic compositionsdescribed herein include chemokines, including without limitation,MCP-1, MfP-1.alpha., MIP-1.beta., and RANTES; adhesion molecules, suchas a selectin, e.g., L-selectin, P-selectin and E-selectin; mucin-likemolecules, e.g., CD34, GlyCAM-1 and MadCAM-1; a member of the integrinfamily such as LFA-1, VLA-1, Mac-1 and p150.95; a member of theimmunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2and ICAM-3, CD2 and LFA-3; co-stimulatory molecules such as B7-1, B7-2,CD40 and CD40L; growth factors including vascular growth factor, nervegrowth factor, fibroblast growth factor, epidermal growth factor, PDGF,BL-1, and vascular endothelial growth factor; receptor moleculesincluding Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3,AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; andCaspases, including ICE.

In some embodiments an adjuvant is a particulate and can have acharacteristic of being slowly biodegradable. Care must be taken toensure that that the adjuvant do not form toxic metabolites. Preferably,in some embodiments, such adjuvants can be matrices used are mainlysubstances originating from a body. These include lactic acid polymers,poly-amino acids (proteins), carbohydrates, lipids and biocompatiblepolymers with low toxicity. Combinations of these groups of substancesoriginating from a body or combinations of substances originating from abody and biocompatible polymers can also be used. Lipids are thepreferred substances since they display structures that make thembiodegradable as well as the fact that they are a critical element inall biological membranes.

In one embodiment, the immunogenic compositions as described herein foradministration must be sterile for administration to a subject.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes), or by gammairradiation.

In some embodiments, the immunogenic compositions described hereinfurther comprise pharmaceutical excipients including, but not limited tobiocompatible oils, physiological saline solutions, preservatives,carbohydrate, protein, amino acids, osmotic pressure controlling agents,carrier gases, pH-controlling agents, organic solvents, hydrophobicagents, enzyme inhibitors, water absorbing polymers, surfactants,absorption promoters and anti-oxidative agents. Representative examplesof carbohydrates include soluble sugars such as hydropropyl cellulose,carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid,chitosan, alginate, glucose, xylose, galactose, fructose, maltose,saccharose, dextran, chondroitin sulfate, etc. Representative examplesof proteins include albumin, gelatin, etc. Representative examples ofamino acids include glycine, alanine, glutamic acid, arginine, lysine,and their salts. Such pharmaceutical excipients are well-known in theart.

In some embodiments, the immunogenic MAPS composition is administered incombination with other therapeutic ingredients including, e.g.,γ-interferon, cytokines, chemotherapeutic agents, or anti-inflammatory,or anti-viral agents. In some embodiments, the immunogenic compositionas disclosed herein can be administered with one or more co-stimulatorymolecules and/or adjuvants as disclosed herein.

In some embodiments, the immunogenic composition is administered in apure or substantially pure form, but may be administered as apharmaceutical composition, formulation or preparation. Such formulationcomprises MAPS described herein together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. Other therapeutic ingredients include compounds thatenhance antigen presentation, e.g., gamma interferon, cytokines,chemotherapeutic agents, or anti-inflammatory agents. The formulationscan conveniently be presented in unit dosage form and may be prepared bymethods well known in the pharmaceutical art. For example, Plotkin andMortimer, in VACCINES (2nd ed., W.B. Saunders Co., 1994) describesvaccination of animals or humans to induce an immune response specificfor particular pathogens, as well as methods of preparing antigen,determining a suitable dose of antigen, and assaying for induction of animmune response.

Formulations of the SA-MAPS compositions as disclosed herein suitablefor intravenous, intramuscular, intranasal, oral, sublingual, vaginal,rectal, subcutaneous, or intraperitoneal administration convenientlycomprise sterile aqueous solutions of the active ingredient withsolutions which are preferably isotonic with the blood of the recipient.Such formulations may be conveniently prepared by dissolving solidactive ingredient in water containing physiologically compatiblesubstances such as sodium chloride (e.g., 0.1M-2.0 M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution, and rendering the solution sterile.These may be present in unit or multi-dose containers, for example,sealed ampoules or vials.

Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Formulations for an intranasal delivery are described in U.S. Pat. No.5,427,782; 5,843,451; 6,398,774.

The formulations of the SA-MAPS compositions as disclosed herein canincorporate a stabilizer. Illustrative stabilizers are polyethyleneglycol, proteins, saccharide, amino acids, inorganic acids, and organicacids which may be used either on their own or as admixtures. Two ormore stabilizers may be used in aqueous solutions at the appropriateconcentration and/or pH. The specific osmotic pressure in such aqueoussolution is generally in the range of 0.1-3.0 osmoses, preferably in therange of 0.80-1.2. The pH of the aqueous solution is adjusted to bewithin the range of pH 5.0-9.0, preferably within the range of pH 6-8.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or morenon-ionic surfactants, including but not limited to polyoxyethylenesorbitan fatty acid esters, Polysorbate-80 (Tween 80), Polysorbate-60(Tween 60), Polysorbate-40 (Tween 40) and Polysorbate-20 (Tween 20),polyoxyethylene alkyl ethers, including but not limited to Brij 58, Brij35, as well as others such as Triton X-100; Triton X-114, NP40, Span 85and the Pluronic series of non-ionic surfactants (e. g., Plutonic 121),with preferred components Polysorbate-80 at a concentration from about0.001% to about 2% (with up to about 0.25% being preferred) orPolysorbate-40 at a concentration from about 0.001% to 1% (with up toabout 0.5% being preferred).

In certain embodiments, a formulation of the SA-MAPS compositions asdisclosed herein comprises one or more additional stabilizing agentssuitable for parenteral administration, e.g., a reducing agentcomprising at least one thiol (—SH) group (e.g., cysteine, N-acetylcysteine, reduced glutathione, sodium thioglycolate, thiosulfate,monothioglycerol, or mixtures thereof). Alternatively, or optionally,preservative-containing immunogenic composition formulations of theinvention may be further stabilized by removing oxygen from storagecontainers, protecting the formulation from light (e.g., by using amberglass containers).

Preservative-containing immunogenic composition formulations of theSA-MAPS composition may comprise one or more pharmaceutically acceptablecarriers or excipients, which includes any excipient that does notitself induce an immune response. Suitable excipients include but arenot limited to macromolecules such as proteins, saccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,sucrose (Paoletti et al, 2001, Vaccine, 19:2118), trehalose, lactose andlipid aggregates (such as oil droplets or liposomes). Such carriers arewell known to the skilled artisan. Pharmaceutically acceptableexcipients are discussed, e.g., in Gennaro, 2000, Remington: The Scienceand Practice of Pharmacy, 20.sup.th edition, ISBN:0683306472.

In some embodiments, SA-MAPS compositions as disclosed herein may belyophilized or in aqueous form, i.e. solutions or suspensions. Liquidformulations may advantageously be administered directly from theirpackaged form and are thus ideal for injection without the need forreconstitution in aqueous medium as otherwise required for lyophilizedcompositions of the invention.

When oral preparations are desired, the immunogenic compositions can becombined with typical carriers, such as lactose, sucrose, starch, talcmagnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers.

In some embodiments, the SA-MAPS immunogenic compositions as describedherein can be administered intravenously, intranasally, intramuscularly,subcutaneously, intraperitoneally, sublingually, vaginal, rectal ororally. In some embodiments, the route of administration is oral,intranasal, subcutaneous, or intramuscular. In some embodiments, theroute of administration is intranasal administration.

Vaccination can be conducted by conventional methods. For example, aSA-MAPS immunogenic composition as disclosed herein can be used in asuitable diluent such as saline or water, or complete or incompleteadjuvants. The immunogenic composition can be administered by any routeappropriate for eliciting an immune response. The SA-MAPS immunogeniccomposition can be administered once or at periodic intervals until animmune response is elicited. Immune responses can be detected by avariety of methods known to those skilled in the art, including but notlimited to, antibody production, cytotoxicity assay, proliferation assayand cytokine release assays. For example, samples of blood can be drawnfrom the immunized mammal, and analyzed for the presence of antibodiesagainst the antigens of the immunogenic composition by ELISA (see deBoer et. al., 115 Arch Virol. 147 (1990) and the titer of theseantibodies can be determined by methods known in the art.

The precise dose of the SA-MAPS to be employed in the formulation willalso depend on the route of administration and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. For example, a range of 25 μg-900 μg total protein can beadministered monthly for three months.

Packaging and Dosage Forms

In some embodiments, the SA-MAPS immunogenic compositions as disclosedherein may be packaged in unit dose or multi-dose form (e.g. 2 doses, 4doses, or more). For multi-dose forms, vials are typically but notnecessarily preferred over pre-filled syringes. Suitable multi-doseformats include but are not limited to: 2 to 10 doses per container at0.1 to 2 mL per dose. In certain embodiments, the dose is a 0.5 mL dose.See, e.g., International Patent Application WO2007/127668, which isincorporated by reference herein.

In some embodiments, the SA-MAPS immunogenic compositions as disclosedherein can be presented in vials or other suitable storage containers,or may be presented in pre-filled delivery devices, e.g., single ormultiple component syringes, which may be supplied with or withoutneedles. A syringe typically but need not necessarily contains a singledose of the preservative-containing immunogenic composition of theinvention, although multi-dose, pre-filled syringes are also envisioned.Likewise, a vial may include a single dose but may alternatively includemultiple doses.

Effective dosage volumes can be routinely established, but a typicaldose of the composition for injection has a volume of 0.5 mL. In certainembodiments, the dose is formulated for administration to a humansubject. In certain embodiments, the dose is formulated foradministration to an adult, teen, adolescent, toddler or infant (i.e.,no more than one year old) human subject and may in preferredembodiments be administered by injection.

Ultimately, the attending physician will decide the amount of theSA-MAPS immunogenic composition or vaccine composition to administer toparticular individuals. As with all immunogenic compositions orvaccines, the immunologically effective amounts of the immunogens (e.g.,the immunogenic polysaccharide and the SA antigens) must be determinedempirically. Factors to be considered include the immunogenicity of thecomposition as a whole (e.g., it is important to note that the SAantigens induce a greater immune response when present in a SA-MAPScomplex as compared to the mixture of the SA antigens alone (notcomplexed), the presence of an adjuvant or co-stimulant as disclosedherein, routes of administrations and the number of immunizing dosagesto be administered. Such factors are known in the vaccine art and it iswell within the skill of immunologists to make such determinationswithout undue experimentation.

Liquid immunogenic compositions of the SA-MAPS immunogenic compositionsas disclosed herein are also suitable for reconstituting otherimmunogenic compositions which are presented in lyophilized form. Wherean immunogenic composition is to be used for such extemporaneousreconstitution, in some embodiment, the present invention provides a kitwith two or more vials, two or more ready-filled syringes, or one ormore of each, with the contents of the syringe being used toreconstitute the contents of the vial prior to injection, or vice versa.

Alternatively, in some embodiments, the SA-MAPS immunogenic compositionsas disclosed herein may be lyophilized and reconstituted, e.g., usingone of a multitude of methods for freeze drying well known in the art toform dry, regular shaped (e.g., spherical) particles, such asmicropellets or microspheres, having particle characteristics such asmean diameter sizes that may be selected and controlled by varying theexact methods used to prepare them. In some embodiments, the SA-MAPSimmunogenic compositions as disclosed herein may further comprise anadjuvant which may optionally be prepared with or contained in separatedry, regular shaped (e.g., spherical) particles such as micropellets ormicrospheres. In some embodiments, the SA-MAPS immunogenic compositionsas disclosed herein are present in a kit comprising a first componentthat includes a stabilized, dry SA-MAPS immunogenic composition asdisclosed herein, optionally further comprising one or morepreservatives, and a second component comprising a sterile, aqueoussolution for reconstitution of the first component. In certainembodiments, the aqueous solution comprises one or more preservatives,and may optionally comprise at least one adjuvant (see, e.g.,WO2009/109550 (incorporated herein by reference).

In yet another embodiment, a container of the multi-dose format isselected from one or more of the group consisting of, but not limitedto, general laboratory glassware, flasks, beakers, graduated cylinders,fermentors, bioreactors, tubings, pipes, bags, jars, vials, vialclosures (e.g., a rubber stopper, a screw on cap), ampoules, syringes,dual or multi-chamber syringes, syringe stoppers, syringe plungers,rubber closures, plastic closures, glass closures, cartridges anddisposable pens and the like. The container of the present invention isnot limited by material of manufacture, and includes materials such asglass, metals (e.g., steel, stainless steel, aluminum, etc.) andpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In a particular embodiment, the container of the format is a 5 mL SchottType I glass vial with a butyl stopper. The skilled artisan willappreciate that the format set forth above is by no means an exhaustivelist, but merely serve as guidance to the artisan with respect to thevariety of formats available for the present invention. Additionalformats contemplated for use in the present invention may be found inpublished catalogues from laboratory equipment vendors and manufacturerssuch as United States Plastic Corp. (Lima, Ohio), VWR.

Kits

The present invention also provides for kits for producing a SA-MAPSimmunogenic composition as disclosed herein which is useful for aninvestigator to tailor an immunogenic composition with their preferredSA antigens, e.g., for research purposes to assess the effect of a SAantigen, or a combination of SA antigens on immune response. Such kitscan be prepared from readily available materials and reagents. Forexample, such kits can comprise any one or more of the followingmaterials: a container comprising an immunogenic polysaccharide,cross-linked with a plurality of first affinity molecules; and acontainer comprising a complementary affinity molecule which associateswith the first affinity molecule, wherein the complementary affinitymolecule associates with a SA antigen.

In another embodiment, the kit can comprise a container comprising animmunogenic polysaccharide, a container comprising a plurality of firstaffinity molecules, and a container comprising a cross-linking reagentfor cross-linking the first affinity molecules to the immunogenicpolysaccharide.

In some embodiments, the kit further comprises a means to attach thecomplementary affinity molecule to the antigen, where the means can beby a cross-linking reagent or by some intermediary fusion protein. Insome embodiments, the kit can comprise at least one co-stimulationfactor which can be added to the polymer. In some embodiments, the kitcomprises a cross-linking reagent, for example, but not limited to, CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodiumcyanoborohydride; cyanogen bromide; ammonium bicarbonate/iodoacetic acidfor linking the co-factor to the polymer.

A variety of kits and components can be prepared for use in the methodsdescribed herein, depending upon the intended use of the kit, theparticular target antigen and the needs of the user.

In one embodiment, a SA-MAPS immunogenic composition or vaccinecomposition as described herein, when administered to mice, can provokean immune response that prevents a disease symptom in at least 20% ofanimals challenged with 5 LD₅₀ of the immunogenic composition comprisingantigens to which the disease symptom is prevented. Methods ofvaccination and challenging an immunized animal are known to one skilledin the art. For example, a 10 μg aliquot of an immunogenic compositionor vaccine composition as disclosed herein can be prepared in 100 μl PBSand/or with addition of alum adjuvants or incomplete Freund's adjuvantand injected subcutaneously per mouse per vaccination. Alternatively,parenteral, intraperitoneal and footpad injections can be used. Volumesof footpad injections are reduced to 50 μl. Mice can be immunized withan immunogenic composition or vaccine composition as disclosed herein onthree separate occasions with several days, e.g., 14 days interval inbetween.

Efficacy of vaccination can be tested by challenge with the pathogen,e.g., S. aureus, or by the method disclosed herein. Seven days after thelast dose of an immunogenic composition, the immunized mice arechallenged intranasally with a pathogenic organism from which theantigen was derived. Ether anaesthetized mice (10 g to 12 g) can beinfected intranasally with 50 μl of PBS-diluted allantoic fluidcontaining 5 LD₅₀ of the pathogenic organism. Protection can be measuredby monitoring animal survival and body weight, which is assessedthroughout an observation period of 21 days. Severely affected mice areeuthanized. One LD₅₀ of A/Mallard/Pennsylvania/10218/84 is equal to100-1000 the Tissue Culture Infectious Dose50 (TCID50) assay.

Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

The term “immunogenic” as used herein means an ability of an antigen (oran epitope of the antigen), such as a bacterial capsular polysaccharideor a conjugate immunogenic composition comprising the bacterial capsularpolysaccharide and a polypeptide or peptide antigen, to elicit an immuneresponse in a host such as a mammal, either humorally or cellularlymediated, or both.

The term “immunogenic composition” used herein is defined as acomposition capable of eliciting an immune response, such as an antibodyor cellular immune response, or both, when administered to a subject.The immunogenic compositions as disclosed herein may or may not beimmunoprotective or therapeutic. When the immunogenic compositions asdisclosed herein prevent, ameliorate, palliate or eliminate disease fromthe subject, then the immunogenic composition may optionally be referredto as a vaccine. As used herein, however, the term immunogeniccomposition is not intended to be limited to vaccines.

Accordingly, an “immunogenic composition” as used herein means anyimmunogenic polysaccharide conjugated to one or more first affinitymolecules, where the first affinity molecule is bound to a complementaryaffinity molecule that is fused to, or otherwise attached to at leastone S. aureus peptide or polypeptide antigen, whereby both theimmunogenic polysaccharide and the S. aureus peptide or polypeptideantigen, each, serve as antigens or antigenic determinant (i.e.,epitopes) of the immunogenic composition to elicit an immune response.That is, the immunogenic composition induces a more robust immuneresponse than each of the components alone (i.e., the immunogenicpolysaccharide alone, or one or more of the S. aureus peptide orpolypeptide antigens alone (i.e., a mixture of one or more of the S.aureus peptide or polypeptide antigens that are not in a complex orconjugated to the polysaccharide). The immunogenic composition may serveto sensitize the host by the presentation of one or more of the S.aureus peptide or polypeptide antigens in association with MHC moleculesat a cell surface. In addition, antigen-specific T-cells or antibodiescan be generated to allow for the future protection of an immunizedhost. Immunogenic composition thus can protect the host from one or moresymptoms associated with infection by the S. aureus, or may protect thehost from death due to the infection with S. aureus. In someembodiments, the SA-MAPS immunogenic compositions as disclosed hereincan also be used to generate polyclonal or monoclonal antibodies, whichmay be used to confer passive immunity to a subject. In someembodiments, the SA-MAPS immunogenic compositions as disclosed hereincan also be used to generate antibodies that are functional as measuredby the killing of bacteria in either an animal efficacy model or via anopsonophagocytic killing assay.

The term “antigen” generally refers to a biological molecule, usually aprotein or polypeptide, peptide, polysaccharide or conjugate in animmunogenic composition, or immunogenic substance that can stimulate theproduction of antibodies or T-cell responses, or both, in an animal,including compositions that are injected or absorbed into an animal. Theimmune response may be generated to the whole molecule (i.e., such asthe SA-MAPS immunogenic composition, or to the whole immunogenicpolysaccharide, or the whole peptide or polypeptide antigen), or to avarious portions of the molecule (e.g., an epitope or hapten within apart of the SA-MAPS immunogenic composition, or to the whole immunogenicpolysaccharide, or the whole peptide or polypeptide antigen). The termmay be used to refer to an individual molecule or to a homogeneous orheterogeneous population of antigenic molecules. An antigen isrecognized by antibodies, T-cell receptors or other elements of specifichumoral and/or cellular immunity. The term “antigen” also includes allrelated antigenic epitopes. Epitopes of a given antigen can beidentified using any number of epitope mapping techniques, well known inthe art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by, e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715; each ofwhich is incorporated herein by reference as if set forth in itsentirety. Similarly, conformational epitopes may be identified bydetermining spatial conformation of amino acids such as by, e.g., x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, supra. Furthermore, for purposes of thepresent invention, “antigen” also can be used to refer to a protein thatincludes modifications, such as deletions, additions and substitutions(generally conservative in nature, but they may be non-conservative), tothe native sequence, so long as the protein maintains the ability toelicit an immunological response. These modifications may be deliberate,as through site-directed mutagenesis, or through particular syntheticprocedures, or through a genetic engineering approach, or may beaccidental, such as through mutations of hosts, which produce theantigens. Furthermore, the antigen can be derived, obtained, or isolatedfrom a microbe, e.g., a bacterium, or can be a whole organism.Similarly, an oligonucleotide polynucleotide, which expresses anantigen, such as in nucleic acid immunization applications, is alsoincluded in the definition. Synthetic antigens are also included, e.g.,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens (Bergmann et al. (1993) Eur. J. Immunol. 23:2777 2781;Bergmann et al. (1996) J. Immunol. 157:3242-3249; Suhrbier (1997)Immunol. Cell Biol. 75:402 408; Gardner et al. (1998) 12th World AIDSConference, Geneva, Switzerland, Jun. 28 to Jul. 3, 1998). In someembodiments, an antigen is a peptide or a polypeptide, e.g., a S. aureuspeptide or a polypeptide, or immunogenic polysaccharide and in otherembodiments, it can be any chemical or moiety, e.g., a carbohydrate,that elicits an immune response directed against the substance.

An “immune response” to an antigen or immunogenic composition is thedevelopment in a subject of a humoral and/or a cell-mediated immuneresponse to molecules present in the antigen or vaccine composition ofinterest. For purposes of the present invention, a “humoral immuneresponse” is an antibody-mediated immune response and involves theinduction and generation of antibodies that recognize and bind with someaffinity for the antigen in the immunogenic composition of theinvention, while a “cell-mediated immune response” is one mediated byT-cells and/or other white blood cells. A “cell-mediated immuneresponse” is elicited by the presentation of antigenic epitopes inassociation with Class I or Class II molecules of the majorhistocompatibility complex (MHC), CD1 or other non-classical MHC-likemolecules. This activates antigen-specific CD4+T helper cells or CD8+cytotoxic lymphocyte cells (“CTLs”). CTLs have specificity for peptideantigens that are presented in association with proteins encoded byclassical or non-classical MHCs and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide or other antigens in associationwith classical or non-classical MHC molecules on their surface. A“cell-mediated immune response” also refers to the production ofcytokines, chemokines and other such molecules produced by activatedT-cells and/or other white blood cells, including those derived fromCD4+ and CD8+ T-cells. The ability of a particular antigen orcomposition to stimulate a cell-mediated immunological response may bedetermined by a number of assays, such as by lymphoproliferation(lymphocyte activation) assays, CTL cytotoxic cell assays, by assayingfor T-lymphocytes specific for the antigen in a sensitized subject, orby measurement of cytokine production by T cells in response tore-stimulation with antigen. Such assays are well known in the art. See,e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al.(1994) Eur. J. Immunol. 24:2369-2376.

The term “treatment” (including variations thereof, e.g., “treat” or“treated”) as used herein means any one or more of the following: (i)the prevention of infection or re-infection, as in a traditionalvaccine, (ii) the reduction in the severity of, or, in the eliminationof symptoms, and (iii) the substantial or complete elimination of thepathogen or disorder in question. Hence, treatment may be effectedprophylactically (prior to infection) or therapeutically (followinginfection). In the present invention, prophylactic treatment is thepreferred mode. According to a particular embodiment of the presentinvention, compositions and methods are provided that treat, includingprophylactically and/or therapeutically immunize, a host animal againsta microbial infection (e.g., a bacterium such as Staphylococcus). Themethods of the present invention are useful for conferring prophylacticand/or therapeutic immunity to a subject. The methods of the presentinvention can also be practiced on subjects for biomedical researchapplications.

The term “mammal” as used herein means a human or non-human animal. Moreparticularly, mammal refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports and petcompanion animals such as a household pet and other domesticated animalincluding, but not limited to, cattle, sheep, ferrets, swine, horses,rabbits, goats, dogs, cats, and the like. In some embodiments, acompanion animal is a dog or cat. Preferably, the mammal is human.

The term an “immunogenic amount,” and “immunologically effectiveamount,” both of which are used interchangeably herein, refers to theamount of the antigen or immunogenic composition sufficient to elicit animmune response, either a cellular (T-cell) or humoral (B-cell orantibody) response, or both, as measured by standard assays known to oneskilled in the art. The “immunogenic amount” of a particular immunogeniccomposition is generally dosed based on total immunogenic polysaccharideand attached or associated SA peptide or polypeptide antigens. Forexample, an SA-MAPS immunogenic composition as disclosed herein willhave at least about 80% or more of, e.g., a serotype 5 or 8 capsularpolysaccharide with attached SA-antigens via the affinity binding pair.Accordingly, in some embodiments, a SA-MAPS immunogenic composition asdisclosed herein can have 20%, or less, free immunogenic (e.g. CP5 orCP8 or a CP5/CP8 conjugate) polysaccharide, and as such, a 100 mcg dosecan have about 80 mcg of immunogenic polysaccharide-antigen SA-MAPScomplex and about 20 mcg, or less, of a non-conjugated immunogenicpolysaccharide. In some embodiments, the dose of the SA-antigensassociated with the immunogenic polysaccharide is important andconsidered when calculating the dose of a SA-MAPS composition toadminister to a subject. The amount of SA-MAPS complex can varydepending upon the number and types of the attached SA antigens, theimmunogenic polysaccharide (e.g., the staphylococcal serotype) as wellas any associated co-stimulants as disclosed herein, as well as route ofadministration, subject and disease to be treated. Generally, eachSA-MAPS dose will comprise 0.1 to 100 mcg of an immunogenicpolysaccharide and attached SA antigens, particularly 0.1 to 10 mcg, andmore particularly 1 to 10 mcg.

The amount of a SA-MAPS immunogenic composition as disclosed herein canvary depending upon the staphylococcal serotype. Generally, each dosewill comprise 0.1 to 100 mcg of immunogenic polysaccharide, particularly0.1 to 10 mcg, and more particularly 1 to 10 mcg. The “immunogenicamount” of the different polysaccharide components in the immunogeniccomposition, may diverge and each may comprise 1 mcg, 2 mcg, 3 mcg, 4mcg, 6 mcg, 6 mcg, 7 mcg, 8 mcg, 9 mcg, 10 mcg, 15 mcg, 20 mcg, 30 mcg,40 mcg, 50 mcg, 60 mcg, 70 mcg, 80 mcg, 90 mcg, or about 100 mcg of anyparticular polysaccharide antigen.

S. aureus “invasive disease” is the isolation of bacteria from anormally sterile site, where there is associated clinical signs/symptomsof disease. Normally sterile body sites include blood, CSF, pleuralfluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone,internal body site (lymph node, brain, heart, liver, spleen, vitreousfluid, kidney, pancreas, ovary) or other normally sterile sites.Clinical conditions characterizing invasive diseases include bacteremia,pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock andmore.

The term “associates” as used herein refers to the linkage of two ormore molecules by non-covalent or covalent bonds. In some embodiments,where linking of two or more molecules occurs by a covalent bond, thetwo or more molecules can be fused together, or cross-linked together.In some embodiments, where linking of two or more molecules occurs by anon-covalent bond, the two or more molecules can form a complex.

The term “complex” as used herein refers to a collection of two or moremolecules, connected spatially by means other than a covalentinteraction; for example, they can be connected by electrostaticinteractions, hydrogen bound or by hydrophobic interactions (i.e., vander Waals forces).

The term “cross-linked” as used herein refers to a covalent bond formedbetween a polymer chain and a second molecule. The term “cross-linkingreagent” refers to an entity or agent which is an intermediate moleculeto catalyze the covalent linkage of a polymer with an entity, e.g.,first affinity molecule or co-stimulatory factor.

As used herein, the term “fused” means that at least one protein orpeptide is physically associated with a second protein or peptide. Insome embodiments, fusion is typically a covalent linkage, however, othertypes of linkages are encompassed in the term “fused” include, forexample, linkage via an electrostatic interaction, or a hydrophobicinteraction and the like. Covalent linkage can encompass linkage as afusion protein or chemically coupled linkage, for example via adisulfide bound formed between two cysteine residues.

As used herein, the term “fusion polypeptide” or “fusion protein” meansa protein created by joining two or more polypeptide sequences together.The fusion polypeptides encompassed in this invention includetranslation products of a chimeric gene construct that joins the DNAsequences encoding one or more antigens, or fragments or mutantsthereof, with the DNA sequence encoding a second polypeptide to form asingle open-reading frame. In other words, a “fusion polypeptide” or“fusion protein” is a recombinant protein of two or more proteins whichare joined by a peptide bond or via several peptides. In someembodiments, the second protein to which the antigens are fused to is acomplementary affinity molecule which is capable of interacting with afirst affinity molecule of the complementary affinity pair.

The terms “polypeptide” and “protein” may be used interchangeably torefer to a polymer of amino acid residues linked by peptide bonds, andfor the purposes of the claimed invention, have a typical minimum lengthof at least 25 amino acids. The term “polypeptide” and “protein” canencompass a multimeric protein, e.g., a protein containing more than onedomain or subunit. The term “peptide” as used herein refers to asequence of peptide bond-linked amino acids containing less than 25amino acids, e.g., between about 4 amino acids and 25 amino acids inlength. Proteins and peptides can be composed of linearly arranged aminoacids linked by peptide bonds, whether produced biologically,recombinantly, or synthetically and whether composed of naturallyoccurring or non-naturally occurring amino acids, are included withinthis definition. Both full-length proteins and fragments thereof greaterthan 25 amino acids are encompassed by the definition of protein. Theterms also include polypeptides that have co-translational (e.g., signalpeptide cleavage) and post-translational modifications of thepolypeptide, such as, for example, disulfide-bond formation,glycosylation, acetylation, phosphorylation, lipidation, proteolyticcleavage (e.g., cleavage by metalloproteases), and the like.Furthermore, as used herein, a “polypeptide” refers to a protein thatincludes modifications, such as deletions, additions, and substitutions(generally conservative in nature as would be known to a person in theart) to the native sequence, as long as the protein maintains thedesired activity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods.

By “signal sequence” is meant a nucleic acid sequence which, whenoperably linked to a nucleic acid molecule, facilitates secretion of theproduct (e.g., protein or peptide) encoded by the nucleic acid molecule.In some embodiments, the signal sequence is preferably located 5′ to thenucleic acid molecule.

As used herein, the term “N-glycosylated” or “N-glycosylation” refers tothe covalent attachment of a sugar moiety to asparagine residues in apolypeptide. Sugar moieties can include but are not limited to glucose,mannose, and N-acetylglucosamine. Modifications of the glycans are alsoincluded, e.g., siaylation.

An “antigen presenting cell” or “APC” is a cell that expresses the MajorHistocompatibility complex (MHC) molecules and can display foreignantigen complexed with MHC on its surface. Examples of antigenpresenting cells are dendritic cells, macrophages, B-cells, fibroblasts(skin), thymic epithelial cells, thyroid epithelial cells, glial cells(brain), pancreatic beta cells, and vascular endothelial cells.

The term “functional portion” or “functional fragment” as used in thecontext of a “functional portion of an antigen” refers to a portion ofthe antigen or antigen polypeptide that mediates the same effect as thefull antigen moiety, e.g., elicits an immune response in a subject, ormediates an association with other molecule, e.g., comprises at least onepitope.

A “portion” of a target antigen as that term is used herein will be atleast 3 amino acids in length, and can be, for example, at least 6, atleast 8, at least 10, at least 14, at least 16, at least 17, at least18, at least 19, at least 20 or at least 25 amino acids or greater,inclusive.

The terms “Cytotoxic T Lymphocyte” or “CTL” refers to lymphocytes whichinduce death via apoptosis or other mechanisms in targeted cells. CTLsform antigen-specific conjugates with target cells via interaction ofTCRs with processed antigen (Ag) on target cell surfaces, resulting inapoptosis of the targeted cell. Apoptotic bodies are eliminated bymacrophages. The term “CTL response” is used to refer to the primaryimmune response mediated by CTL cells.

The term “cell mediated immunity” or “CMI” as used herein refers to animmune response that does not involve antibodies or complement butrather involves the activation of, for example, macrophages, naturalkiller cells (NK), antigen-specific cytotoxic T-lymphocytes (T-cells),T-helper cells, neutrophils, and the release of various cytokines inresponse to a target antigen. Stated another way, CMI refers to immunecells (such as T cells and other lymphocytes) which bind to the surfaceof other cells that display a target antigen (such as antigen presentingcells (APC)) and trigger a response. The response may involve eitherother lymphocytes and/or any of the other white blood cells (leukocytes)and the release of cytokines. Cellular immunity protects the body by:(i) activating antigen-specific cytotoxic T-lymphocytes (CTLs) that areable to destroy body cells displaying epitopes of foreign antigen ontheir surface, such as virus-infected cells and cells with intracellularbacteria; (2) activating macrophages and NK cells, enabling them todestroy intracellular pathogens; and (3) stimulating cells to secrete avariety of cytokines or chemokines that influence the function of othercells such as T cells, macrophages or neutrophils involved in adaptiveimmune responses and innate immune responses.

The term “immune cell” as used herein refers to any cell which canrelease a cytokine, chemokine or antibody in response to a direct orindirect antigenic stimulation. Included in the term “immune cells”herein are lymphocytes, including natural killer (NK) cells, T-cells(CD4+ and/or CD8+ cells), B-cells, macrophages; leukocytes; dendriticcells; mast cells; monocytes; and any other cell which is capable ofproducing a cytokine or chemokine molecule in response to direct orindirect antigen stimulation. Typically, an immune cell is a lymphocyte,for example a T-cell lymphocyte.

A “protective” immune response refers to the ability of an immunogeniccomposition as disclosed herein to elicit an immune response, eitherhumoral or cell mediated, or both, which serves to protect a subjectfrom an infection. The protection provided need not be absolute, i.e.,the infection need not be totally prevented or eradicated, if there is astatistically significant improvement compared with a control populationof subjects, e.g. infected animals not administered the vaccine orimmunogenic composition. Protection may be limited to mitigating theseverity or rapidity of onset of symptoms of the infection. In general,a “protective immune response” would include the induction of anincrease in antibody levels specific for a particular antigen in atleast 50% of subjects, including some level of measurable functionalantibody responses to each antigen. In particular situations, a“protective immune response” could include the induction of a two-foldincrease in antibody levels or a fourfold increase in antibody levelsspecific for a particular antigen in at least 50% of subjects, includingsome level of measurable functional antibody responses to each antigen.In certain embodiments, opsonising antibodies correlate with aprotective immune response. Thus, protective immune response may beassayed by measuring the percent decrease in the bacterial count in anopsonophagocytosis assay, for instance those described below.Preferably, there is a decrease in bacterial count of at least 10%, 25%,50%, 65%, 75%, 80%, 85%, 90%, 95% or more.

The term “cytokine” as used herein refers to a molecule released from animmune cell in response to stimulation with an antigen. Examples of suchcytokines include, but are not limited to: GM-CSF; IL-1α; IL-1β; IL-2;IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F, orother members of the IL-17 family, IL-22, IL-23, IFN-α; IFN-β; IFN-γ;MIP-1α; MIP-1β; TGF-β; TNFα, or TNFβ. The term “cytokine” does notinclude antibodies.

The term “subject” as used herein refers to any animal in which it isuseful to elicit an immune response. The subject can be a wild,domestic, commercial or companion animal such as a bird or mammal. Thesubject can be a human. Although in one embodiment of the invention itis contemplated that the immunogenic compositions as disclosed hereincan also be suitable for the therapeutic or preventative treatment inhumans, it is also applicable to warm-blooded vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, and non-mammals such as chickens, ducks, or turkeys. Inanother embodiment, the subject is a wild animal, for example a birdsuch as for the diagnosis of avian flu. In some embodiments, the subjectis an experimental animal or animal substitute as a disease model. Thesubject may be a subject in need of veterinary treatment, whereeliciting an immune response to an antigen is useful to prevent adisease and/or to control the spread of a disease, for example SIV,STL1, SFV, or in the case of live-stock, hoof and mouth disease, or inthe case of birds Marek's disease or avian influenza, and other suchdiseases.

As used herein, the term “pathogen” refers to an organism or moleculethat causes a disease or disorder in a subject. For example, pathogensinclude but are not limited to viruses, fungi, bacteria, parasites, andother infectious organisms or molecules therefrom, as well astaxonomically related macroscopic organisms within the categories algae,fungi, yeast, protozoa, or the like.

The term “wild type” refers to the naturally-occurring, normalpolynucleotide sequence encoding a protein, or a portion thereof, orprotein sequence, or portion thereof, respectively, as it normallyexists in vivo.

The term “mutant” refers to an organism or cell with any change in itsgenetic material, in particular a change (i.e., deletion, substitution,addition, or alteration) relative to a wild-type polynucleotide sequenceor any change relative to a wild-type protein sequence. The term“variant” may be used interchangeably with “mutant”. Although it isoften assumed that a change in the genetic material results in a changeof the function of the protein, the terms “mutant” and “variant” referto a change in the sequence of a wild-type protein regardless of whetherthat change alters the function of the protein (e.g., increases,decreases, imparts a new function), or whether that change has no effecton the function of the protein (e.g., the mutation or variation issilent).

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. The term “pharmaceutically acceptable carriers” excludestissue culture medium. Exemplary pharmaceutically acceptable saltsinclude but are not limited to mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like, andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Pharmaceutically acceptable carriers arewell-known in the art.

It will be appreciated that proteins or polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, can be modified in a given polypeptide, eitherby natural processes such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Known modifications which can be present inpolypeptides of the present invention include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa polynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formulation, gamma-carboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicate that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment are identical, with appropriate nucleotide insertions ordeletions or amino-acid insertions or deletions, typically in at least70% of the nucleotides of the nucleotides for high homology. For apolypeptide, there should be at least 30% of amino acid identity in thepolypeptide, or at least 50% for higher homology. The term “homolog” or“homologous” as used herein also refers to homology with respect tostructure. Determination of homologs of genes or polypeptides can beeasily ascertained by the skilled artisan. When in the context with adefined percentage, the defined percentage homology means at least thatpercentage of amino acid similarity. For example, 85% homology refers toat least 85% of amino acid similarity.

As used herein, the term “heterologous” reference to nucleic acidsequences, proteins or polypeptides mean that these molecules are notnaturally occurring in that cell. For example, the nucleic acid sequencecoding for a fusion antigen polypeptide described herein that isinserted into a cell, e.g. in the context of a protein expressionvector, is a heterologous nucleic acid sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Where necessary or desired, optimalalignment of sequences for comparison can be conducted by any variety ofapproaches, as these are well-known in the art.

The term “variant” as used herein may refer to a polypeptide or nucleicacid that differs from the naturally occurring polypeptide or nucleicacid by one or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or morespecific functions or biological activities of the naturally occurringmolecule. Amino acid substitutions include alterations in which an aminoacid is replaced with a different naturally-occurring or anon-conventional amino acid residue. Such substitutions may beclassified as “conservative,” in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality or size. Substitutions encompassed by variants asdescribed herein may also be “non conservative,” in which an amino acidresidue which is present in a peptide is substituted with an amino acidhaving different properties (e.g., substituting a charged or hydrophobicamino acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. Also encompassed within the term “variant,” when used withreference to a polynucleotide or polypeptide, are variations in primary,secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide).

The term “substantially similar,” when used in reference to a variant ofan antigen or a functional derivative of an antigen as compared to theoriginal antigen means that a particular subject sequence varies fromthe sequence of the antigen polypeptide by one or more substitutions,deletions, or additions, but retains at least 50%, or higher, e.g., atleast 60%, 70%, 80%, 90% or more, inclusive, of the function of theantigen to elicit an immune response in a subject. In determiningpolynucleotide sequences, all subject polynucleotide sequences capableof encoding substantially similar amino acid sequences are considered tobe substantially similar to a reference polynucleotide sequence,regardless of differences in codon sequence. A nucleotide sequence is“substantially similar” to a given antigen nucleic acid sequence if: (a)the nucleotide sequence hybridizes to the coding regions of the nativeantigen sequence, or (b) the nucleotide sequence is capable ofhybridization to nucleotide sequence of the native antigen undermoderately stringent conditions and has biological activity similar tothe native antigen protein; or (c) the nucleotide sequences aredegenerate as a result of the genetic code relative to the nucleotidesequences defined in (a) or (b). Substantially similar proteins willtypically be greater than about 80% similar to the correspondingsequence of the native protein.

Variants can include conservative or non-conservative amino acidchanges, as described below. Polynucleotide changes can result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. Variants can also includeinsertions, deletions or substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of omithine which donot normally occur in human proteins. “Conservative amino acidsubstitutions” result from replacing one amino acid with another thathas similar structural and/or chemical properties. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. For example, the following six groups each containamino acids that are conservative substitutions for one another: (1)Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamicacid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine(K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See. e.g.,Creighton, PROTEINS (W.H. Freeman & Co., 1984).

The choice of conservative amino acids may be selected based on thelocation of the amino acid to be substituted in the peptide, for exampleif the amino acid is on the exterior of the peptide and exposed tosolvents, or on the interior and not exposed to solvents. Selection ofsuch conservative amino acid substitutions is within the skill of one ofordinary skill in the art. Accordingly, one can select conservativeamino acid substitutions suitable for amino acids on the exterior of aprotein or peptide (i.e. amino acids exposed to a solvent). Thesesubstitutions include, but are not limited to the following:substitution of Y with F, T with S or K, P with A, E with D or Q, N withD or G, R with K, G with N or A, T with S or K, D with N or E, I with Lor V, F with Y, S with T or A, R with K, G with N or A, K with R, A withS, K or P.

Alternatively, one can also select conservative amino acid substitutionssuitable for amino acids on the interior of a protein or peptide (i.e.,the amino acids are not exposed to a solvent). For example, one can usethe following conservative substitutions: where Y is substituted with F,T with A or S, I with L or V, W with Y, M with L, N with D, G with A, Twith A or S, D with N, I with L or V, F with Y or L, S with A or T and Awith S, G, T or V. In some embodiments, LF polypeptides includingnon-conservative amino acid substitutions are also encompassed withinthe term “variants.” As used herein, the term “non-conservative”substitution refers to substituting an amino acid residue for adifferent amino acid residue that has different chemical properties.Non-limiting examples of non-conservative substitutions include asparticacid (D) being replaced with glycine (G); asparagine (N) being replacedwith lysine (K); and alanine (A) being replaced with arginine (R).

The term “derivative” as used herein refers to proteins or peptideswhich have been chemically modified, for example by ubiquitination,labeling, pegylation (derivatization with polyethylene glycol) oraddition of other molecules. A molecule is also a “derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties can improve themolecule's solubility, absorption, biological half-life, etc. Themoieties can alternatively decrease the toxicity of the molecule, oreliminate or attenuate an undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in REMINGTON'SPHARMACEUTICAL SCIENCES (21st ed., Tory, ed., Lippincott Williams &Wilkins, Baltimore, Md., 2006).

The term “functional” when used in conjunction with “derivative” or“variant” refers to a protein molecule which possesses a biologicalactivity that is substantially similar to a biological activity of theentity or molecule of which it is a derivative or variant.“Substantially similar” in this context means that the biologicalactivity, e.g., antigenicity of a polypeptide, is at least 50% as activeas a reference, e.g., a corresponding wild-type polypeptide, e.g., atleast 60% as active, 70% as active, 80% as active, 90% as active, 95% asactive, 100% as active or even higher (i.e., the variant or derivativehas greater activity than the wild-type), e.g., 110% as active, 120% asactive, or more, inclusive.

The term “recombinant” when used to describe a nucleic acid molecule,means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/orsynthetic origin, which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide sequences withwhich it is associated in nature. The term recombinant as used withrespect to a peptide, polypeptide, protein, or recombinant fusionprotein, means a polypeptide produced by expression from a recombinantpolynucleotide. The term recombinant as used with respect to a host cellmeans a host cell into which a recombinant polynucleotide has beenintroduced. Recombinant is also used herein to refer to, with referenceto material (e.g., a cell, a nucleic acid, a protein, or a vector) thatthe material has been modified by the introduction of a heterologousmaterial (e.g., a cell, a nucleic acid, a protein, or a vector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of a heterologous nucleic acid towhich it has been linked to a host cell; a plasmid is a species of thegenus encompassed by the term “vector.” The term “vector” typicallyrefers to a nucleic acid sequence containing an origin of replicationand other entities necessary for replication and/or maintenance in ahost cell. Vectors capable of directing the expression of genes and/ornucleic acid sequence to which they are operatively linked are referredto herein as “expression vectors”. In general, expression vectors ofutility are often in the form of “plasmids” which refer to circulardouble stranded DNA molecules which, in their vector form are not boundto the chromosome, and typically comprise entities for stable ortransient expression or the encoded DNA. Other expression vectors thatcan be used in the methods as disclosed herein include, but are notlimited to plasmids, episomes, bacterial artificial chromosomes, yeastartificial chromosomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the particular cell. A vector can be a DNA or RNA vector. Other formsof expression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example self replicatingextrachromosomal vectors or vectors which integrates into a host genome.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked.

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease by a statistically significant amount relative to areference. For avoidance of doubt, “reduced” means statisticallysignificant decrease of at least 10% as compared to a reference level,for example a decrease by at least 20%, at least 30%, at least 40%, atleast t 50%, or least 60%, or least 70%, or least 80%, at least 90% ormore, up to and including a 100% decrease (i.e., absent level ascompared to a reference sample), or any decrease between 10-100% ascompared to a reference level, as that term is defined herein.

The term “low” as used herein generally means lower by a staticallysignificant amount; for the avoidance of doubt, “low” means astatistically significant value at least 10% lower than a referencelevel, for example a value at least 20% lower than a reference level, atleast 30% lower than a reference level, at least 40% lower than areference level, at least 50% lower than a reference level, at least 60%lower than a reference level, at least 70% lower than a reference level,at least 80% lower than a reference level, at least 90% lower than areference level, up to and including 100% lower than a reference level(i.e., absent level as compared to a reference sample).

The terms “increased” or “increase” as used herein generally mean anincrease by a statically significant amount; such as a statisticallysignificant increase of at least 10% as compared to a reference level,including an increase of at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100% or more, inclusive, including, for example at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-foldincrease or greater as compared to a reference level, as that term isdefined herein.

The term “high” as used herein generally means a higher by a staticallysignificant amount relative to a reference; such as a statisticallysignificant value at least 10% higher than a reference level, forexample at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher, inclusive, suchas at least 2-fold higher, at least 3-fold higher, at least 4-foldhigher, at least 5-fold higher, at least 10-fold higher or more, ascompared to a reference level.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity of understandingit will be readily apparent to one of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims. The following is meant to be illustrativeof the present invention; however, the practice of the invention is notlimited or restricted in any way by the examples.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. An immunogenic composition comprising an immunogenic        polysaccharide, at least one S. aureus peptide or polypeptide        antigen, and at least one complementary affinity-molecule pair        comprising: a first affinity molecule associated with the        immunogenic polysaccharide, and a complementary affinity        molecule associated with the at least S. aureus peptide or        polypeptide antigen,    -   wherein the first affinity molecule associates with the        complementary affinity molecule to link the S. aureus peptide or        polypeptide antigen and the immunogenic polysaccharide.    -   2. The immunogenic composition of paragraph 1, wherein at least        one S. aureus peptide or polypeptide antigen is selected from        any of the group comprising: hemolysin (HI), Clumping factor A        (ClfA), Clumping factor B (ClfB), serine-aspirate repeat protein        D (SdrD), serine-aspirate repeat protein E (SdrE), Iron        regulator surface protein A (IsdA), Iron regulator surface        protein B (IsdB), Leukotoxin D (LukD), or Leukotoxin E (LukE).    -   3. The immunogenic composition of paragraph 1, wherein the        immunogenic composition comprises a hemolysin (HI) S. aureus        antigen and at least one additional S. aureus antigen selected        from any of the group comprising: Clumping factor A (ClfA),        Clumping factor B (ClfB), serine-aspirate repeat protein D        (SdrD), serine-aspirate repeat protein E (SdrE), Iron regulator        surface protein A (IsdA), Iron regulator surface protein B        (IsdB), Leukotoxin D (LukD), or Leukotoxin E (LukE).    -   4. The immunogenic composition of paragraph 1, wherein the        immunogenic composition comprises a hemolysin (HI) S. aureus        antigen and at least two or more additional S. aureus antigen        selected from any of the group comprising: Clumping factor A        (ClfA), Clumping factor B (ClfB), serine-aspirate repeat protein        D (SdrD), serine-aspirate repeat protein E (SdrE), Iron        regulator surface protein A (IsdA), Iron regulator surface        protein B (IsdB), Leukotoxin D (LukD), or Leukotoxin E (LukE).    -   5. The immunogenic composition of paragraph 4, wherein the        immunogenic composition comprises a hemolysin α (Hla) antigen,        and a Clumping factor A (ClfA) antigen, and a Clumping factor B        (ClfB) antigen, and a serine-aspirate repeat protein D (SdrD)        antigen, and a Iron regulator surface protein A (IsdA) antigen,        and an Iron regulator surface protein B (IsdB) antigen.    -   6. The immunogenic composition of paragraph 5, wherein the        immunogenic composition comprises S. aureus antigens        Hla209(27-319), ClfA (221-559), ClfB (203-542), SdrD (246-682),        IsdA (47-324) and IsdB (48-447).    -   7. The immunogenic composition of any of paragraphs 1 to 6,        wherein Hl antigen is a α-hemolysin (Hla), a β-hemolysin (Hlb)        or a γ-hemolysin (Hl-gamma) from S. aureus.    -   8. The immunogenic composition of any of paragraphs 1 to 6,        wherein Hl is wildtype Hla (WT Hla) or a Hla with a reduced        hemolytic activity or is a non-hemolytic Hla protein.    -   9. The immunogenic composition of any of paragraphs 1 to 8,        wherein the Hla antigen with a reduced hemolytic activity        comprises amino acids of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID        NO: 17 or SEQ ID NO: 18 or a polypeptide with at least 85%        sequence identity thereto.    -   10. The immunogenic composition of any of paragraphs 1 to 8,        wherein the Hla antigen with a reduced hemolytic activity is        amino acids of SEQ ID NO: 16 or a polypeptide with at least 85%        sequence identity thereto.    -   11. The immunogenic composition of any of paragraphs 1 to 5,        wherein the ClfA antigen comprises at least SEQ ID NO: 3 or a        polypeptide with at least 85% sequence identity to SEQ ID NO: 3.    -   12. The immunogenic composition of any of paragraphs 1 to 5,        wherein the ClfA antigen comprises a fragment of at least 30        amino acids of SEQ ID NO: 2 or a polypeptide of at least 30        amino acids that has at least 85% sequence identity to a portion        of SEQ ID NO: 2.    -   13. The immunogenic composition of any of paragraphs 1 to 5,        wherein the ClfB antigen comprises at least SEQ ID NO: 5 or a        polypeptide with at least 85% sequence identity to SEQ ID NO: 5.    -   14. The immunogenic composition of any of paragraphs 1 to 5,        wherein the ClfB antigen comprises a fragment of at least 30        amino acids of SEQ ID NO: 4 or a polypeptide of at least 30        amino acids that has at least 85% sequence identity to a portion        of SEQ ID NO: 4.    -   15. The immunogenic composition of any of paragraphs 1 to 5,        wherein the SdrD antigen comprises at least SEQ ID NO: 7 or a        polypeptide with at least 85% sequence identity to SEQ ID NO: 7.    -   16. The immunogenic composition of any of paragraphs 1 to 5,        wherein the SdrD antigen comprises a fragment of at least 30        amino acids of SEQ ID NO:6 or a polypeptide of at least 30 amino        acids that has at least 85% sequence identity to a portion of        SEQ ID NO: 6.    -   17. The immunogenic composition of any of paragraphs 1 to 5,        wherein the SdrE antigen comprises a fragment of at least 30        amino acids of SEQ ID NO:8 or a polypeptide of at least 30 amino        acids that has at least 85% sequence identity to a portion of        SEQ ID NO: 8.    -   18. The immunogenic composition of any of paragraphs 1 to 5,        wherein the IsdA antigen comprises at least SEQ ID NO: 11 or a        polypeptide with at least 85% sequence identity to SEQ ID NO:        11.    -   19. The immunogenic composition of any of paragraphs 1 to 5,        wherein the IsdA antigen comprises a fragment of at least 30        amino acids of SEQ ID NO:10 or a polypeptide of at least 30        amino acids that has at least 85% sequence identity to a portion        of SEQ ID NO: 10.    -   20. The immunogenic composition of any of paragraphs 1 to 5,        wherein the IsdB antigen comprises at least SEQ ID NO: 13 or a        polypeptide with at least 85% sequence identity to SEQ ID NO:        13.    -   21. The immunogenic composition of any of paragraphs 1 to 5,        wherein the IsdB antigen comprises a fragment of at least 30        amino acids of SEQ ID NO:12 or a polypeptide of at least 30        amino acids that has at least 85% sequence identity to a portion        of SEQ ID NO: 12.    -   22. The immunogenic composition of paragraph 1, wherein the        first affinity molecule is biotin or a derivative or mimic        molecule thereof.    -   23. The immunogenic composition of paragraph 1, wherein the        first affinity molecule is a biotin derivative, lipoic acid,        HABA (hydroxyazobenzene-benzoic acid) or/and dimethyl-HABA or an        amine-PEG3-biotin ((+)-biotinylation-3-6,        9-trixaundecanediamine).    -   24. The immunogenic composition of paragraph 1, wherein the        complementary affinity molecule is a biotin-binding protein, or        an avidin-like protein.    -   25. The immunogenic composition of paragraph 24, wherein the        avidin-like protein is selected from the group consisting of:        rhizavidin, avidin, streptavidin, or a homologue or derivative        thereof.    -   26. The immunogenic composition of paragraph 25, wherein the        rhizavidin is amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1.    -   27. The immunogenic composition of any of paragraphs 1 to 26,        wherein the S. aureus antigen is a fusion protein comprising        the S. aureus antigen fused to a complementary affinity binding        molecule.    -   28. The immunogenic composition of paragraph 1, wherein the        first affinity molecule is cross-linked to the immunogenic        polysaccharide.    -   29. The immunogenic composition of paragraph 1, wherein the        first affinity molecule is cross-linked to the immunogenic        polysaccharide using a cross-linking reagent selected from any        in the group consisting of: CDAP        (1-cyano-4-dimethylaminopyridinium tetrafluoroborate); EDC        (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride);        sodium cyanoborohydride; cyanogen bromide; and ammonium        bicarbonate/iodoacetic acid.    -   30. The immunogenic composition of paragraph 1, wherein the        first affinity molecule is cross-linked to carboxyl, hydroxyl,        amino, phenoxyl, hemiacetal, and mecapto functional groups of        the immunogenic polysaccharide.    -   31. The immunogenic composition of paragraph 1, wherein the        first affinity molecule is covalently bonded to the immunogenic        polysaccharide.    -   32. The immunogenic composition of paragraph 1, wherein the        first affinity molecule and complementary affinity molecule pair        can be selected from a group consisting of:        biotin/biotin-binding protein, antibody/antigen,        enzyme/substrate, receptor/ligand, metal/metal-binding protein,        carbohydrate/carbohydrate binding protein, lipid/lipid-binding        protein, His tag/His tag-binding substance.    -   33. The immunogenic composition of any of paragraphs 1 to 32,        wherein the antigen is non-covalently attached, or covalently        attached to the complementary affinity molecule.    -   34. The immunogenic composition of any of paragraphs 1 to 27,        wherein a secretion signal peptide is located at the N terminal        of the avidin-like protein.    -   35. The immunogenic composition of any of paragraphs 1 to 34,        wherein the secretion signal sequence comprises at least        MKKIWLALAGLVLAFSASA (SEQ ID NO: 23) or MKKIWLALAGLVLAFSASAAQDP        (SEQ ID NO: 24) or an amino acid sequence having at least 85%        identity thereof.    -   36. The immunogenic composition of any of paragraph 1 to 35,        wherein the immunogenic polysaccharide is purified from living        organisms or is a synthetic immunogenic polysaccharide.    -   37. The immunogenic composition of any of paragraph 1 to 36,        wherein the living organism is selected from the group        consisting of: bacteria, archaea, eukaryotic cells, fungi,        insects, plants, animals, or chimeras thereof.    -   38. The immunogenic composition of any of paragraphs 1 to 37,        further comprising a flexible linker peptide attached to the        antigen, wherein the flexible linker peptide attaches the        antigen to the complementary affinity molecule.    -   39. The immunogenic composition of any of paragraphs 1 to 38,        comprising at least 3 S. aureus peptide or polypeptide antigens.    -   40. The immunogenic composition of any of paragraphs 1 to 39,        comprising at least 5 S. aureus peptide or polypeptide antigens.    -   41. The immunogenic composition of any of paragraphs 1 to 40,        comprising between 2-10 S. aureus peptide or polypeptide        antigens.    -   42. The immunogenic composition of any of paragraphs 1 to 40,        comprising between 10-15 S. aureus peptide or polypeptide        antigens.    -   43. The immunogenic composition of any of paragraphs 1 to 41,        comprising between 15-20 S. aureus peptide or polypeptide        antigens.    -   44. The immunogenic composition of any of paragraphs 1 to 42,        comprising between 20-50 S. aureus peptide or polypeptide        antigens.    -   45. The immunogenic composition of any of paragraphs 1 to 43,        comprising between 50-100 S. aureus peptide or polypeptide        antigens.    -   46. The immunogenic composition of any of paragraphs 1 to 44,        comprising more than 100 S. aureus peptide or polypeptide        antigens.    -   47. The immunogenic composition of any of paragraphs 1 to 45,        wherein the immunogenic polysaccharide is selected from a        polysaccharide from the group consisting of: S. aureus, Vi        polysaccharide, pneumococcal capsular polysaccharides,        pneumococcal cell wall polysaccharide, Haemophilus influenzae        Type b polysaccharide, Meningococcal polysaccharide, 0-antigens        from Gram-negative bacteria and other bacterial capsular or cell        wall polysaccharides.    -   48. The immunogenic composition of any of paragraphs 1 to 46,        wherein the immunogenic polysaccharide is selected from type 1        capsular polysaccharide of Streptococcus pneumoniae, type 5        capsular polysaccharide of S. aureus or type 8 capsular        polysaccharide of S. aureus.    -   49. The immunogenic composition of any of paragraphs 1 to 48,        further comprising at least one co-stimulation factor associated        to the immunogenic polysaccharide.    -   50. The immunogenic composition of any of paragraphs 1 to 49,        wherein the co-stimulation factor is selected from the group        consisting of: Toll like receptor ligand or agonists, NOD ligand        or agonists, or activator/agonists of the inflammasome.    -   51. The immunogenic composition of paragraph 50, wherein the        co-stimulation factor is attached to immunogenic polysaccharide        directly, or via a complementary affinity-molecule pair        comprising: a first affinity molecule which associates with the        immunogenic polysaccharide, and a complementary affinity        molecule which associates with the co-stimulation factor,        wherein the first affinity molecule associates with the        complementary affinity molecule to link the co-stimulatory        factor to the immunogenic polysaccharide.    -   52. The immunogenic composition of paragraph 1, wherein        composition is used to elicit an immune response to S. aureus in        a subject.    -   53. The immunogenic composition of paragraph 52, wherein the        immune response is an antibody or B cell response.    -   54. The immunogenic composition of paragraph 52, wherein the        immune response is an antibody or B cell response and T-cell        response.    -   55. The immunogenic composition of paragraph 52, wherein the        immune response is to at least one immunogenic polysaccharide        and at least one peptide or polypeptide S. aureus antigen.    -   56. The immunogenic composition of paragraph 52, wherein the        immune response is a CD4+ T cell response, including Th1, Th2,        or Th17 or Th22 response, or a CD8+ T cell response, or CD4+ and        CD8+ T cell response.    -   57. The immunogenic composition of paragraph 52, wherein the        immune response is an antibody or B cell response to at least        one antigenic polysaccharide and a CD4+ T cell response,        including Th1, Th2, or Th17 or Th22 response, or a CD8+ T cell        response, or CD4+/CD8+ T cell response to at least one peptide        or polypeptide antigen.    -   58. The immunogenic composition of paragraph 52, wherein the        immune response is an antibody or B cell response to at least        one antigenic polysaccharide, and an antibody or B cell response        and a CD4+ T cell response, including Th1, Th2, Th7 or Th22        responses, or a CD8+ T cell response, or CD4+/CD8+ T cell        response to at least one peptide or polypeptide antigen.    -   59. The immunogenic composition of paragraph 52, wherein the        immune response results in activation of INF-γ, IL-17A or IL-22        producing cells, or INF-γ, IL-17A and IL-22 producing cells.    -   60. The immunogenic composition of paragraph 48, wherein the        immune response is an antibody or B cell response against the S.        aureus antigen which associates with the immunogenic        polysaccharide.    -   61. The immunogenic composition of any of paragraphs 1 to 60,        further comprising at least one adjuvant.    -   62. The immunogenic composition of paragraphs 1 to 61 for use in        a diagnostic for exposure to a pathogen or immune threat.    -   63. The immunogenic composition of paragraphs 1 to 61 for use in        preventing infection by S. aureus.    -   64. The immunogenic composition of paragraphs 1 to 61 for use in        preventing colonization of a subject by S. aureus.    -   65. A method for inducing an immune response in a subject to S.        aureus, comprising administering to the subject a composition of        paragraph 1 to 61.    -   66. A method of vaccinating a mammal against at least one        antigen-bearing pathogen, the method comprising administering an        immunogenic composition of paragraph 1 to 61.    -   67. The method of any of paragraphs 65 or 66, wherein the        subject is a human.    -   68. The method of any of paragraphs 65 or 66, wherein the        subject is an agricultural or non-domestic animal.    -   69. The method of any of paragraphs 65 or 66, wherein the        subject is a domestic animal.    -   70. The method of any of paragraphs 65 or 66, wherein        administration is via subcutaneous, intranasal, intradermal, or        intra muscular injection, or via transdermal skin patch.    -   71. The method of paragraph 65, wherein the immune response is        an antibody or B cell response.    -   72. The method of paragraph 65, wherein the immune response is        an antibody or B cell response and T-cell response.    -   73. The method of paragraph 65, wherein the immune response is        to at least one immunogenic polypeptide and at least one peptide        or polypeptide S. aureus antigen.    -   74. The method of paragraph 65, wherein the immune response is a        CD4+ T cell response, including Th1, Th2, Th17 or Th22 response,        or a CD8+ T cell response, or CD4+ and CD8+ T cell response.    -   75. The method of paragraph 65, wherein the immune response is        an antibody or B cell response to at least one antigenic        polysaccharide and a CD4+ T cell response, including Th1, Th2,        Th17 or Th22 response, or a CD8+ T cell response, or CD4+/CD8+ T        cell response to at least one peptide or polypeptide antigen.    -   76. The method of paragraph 65, wherein the immune response is        an antibody or B cell response to at least one antigenic        polysaccharide, and an antibody or B cell response and a CD4+ T        cell response, including Th1, Th2, Th7 or Th22 response, or a        CD8+ T cell response, or CD4+/CD8+ T cell response to at least        one peptide or polypeptide antigen.    -   77. The method of paragraph 65, wherein the immune response        results in activation of IL-17A or IL-22, INF-γ producing cells,        or IL-17A and IL-22 producing cells.    -   78. The method of paragraph 65, wherein the immune response is        an antibody or B cell response against the S. aureus antigen        which associates with the immunogenic polysaccharide.    -   79. A fusion protein comprising a rhizavidin protein and at        least one S. aureus peptide or polypeptide antigen, wherein the        rhizavidin protein comprises amino acids of SEQ ID NO: 1, or 85%        sequence identity to amino acids of SEQ ID NO: 1, and the S.        aureus peptide or polypeptide comprises a fragment of at least        20 amino acids of a protein selected from any of: haemolysin        (HI), Clumping factor A (ClfA), Clumping factor B (ClfB),        serine-aspirate repeat protein D (SdrD), serine-aspirate repeat        protein E (SdrE), Iron regulator surface protein A (IsdA), Iron        regulator surface protein B (IsdB), Leukoptoxin D (LukD), or        Leukoptoxin E (LukE).    -   80. The fusion protein of paragraph 79, wherein the S. aureus        peptide is selected from any of Hla209(27-319), ClfA (221-559),        ClfB (203-542), SdrD (246-682), IsdA (47-324) and IsdB (48-447).    -   81. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a non-hemolytic variant of a        Hla protein.    -   82. The fusion protein of paragraph 81, wherein the        non-haemolytic variant of a Hla protein comprises at least SEQ        ID NO: 16 or a protein of at least 20 amino acids that has at        least 85% sequence identity to SEQ ID NO: 16.    -   83. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a fragment of at least 20 amino        acids of a Clumping factor A (ClfA) protein.    -   84. The fusion protein of paragraph 83, wherein the ClfA protein        comprises at least SEQ ID NO: 3 or a protein of at least 20        amino acids that has at least 85% sequence identity to SEQ ID        NO: 3.    -   85. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a fragment of at least 20 amino        acids of a Clumping factor B (ClfB) protein.    -   86. The fusion protein of paragraph 85, wherein the ClfB protein        comprises at least SEQ ID NO: 5 or a protein of at least 20        amino acids that has at least 85% sequence identity to SEQ ID        NO: 5.    -   87. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a fragment of at least 20 amino        acids of a serine-aspirate repeat protein D (SdrD) protein.    -   88. The fusion protein of paragraph 87, wherein the SdrD protein        comprises at least SEQ ID NO: 7 or a protein of at least 20        amino acids that has at least 85% sequence identity to SEQ ID        NO: 7.    -   89. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a fragment of at least 20 amino        acids of a serine-aspirate repeat protein D (SdrED) protein.    -   90. The fusion protein of paragraph 89, wherein the SdrE protein        comprises at least SEQ ID NO: 8 or a protein of at least 20        amino acids that has at least 85% sequence identity to SEQ ID        NO: 8.    -   91. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a fragment of at least 20 amino        acids of Iron regulator surface protein A (IsdA), protein.    -   92. The fusion protein of paragraph 91, wherein the IsdA protein        comprises at least SEQ ID NO: 11 or a protein of at least 20        amino acids that has at least 85% sequence identity to SEQ ID        NO: 11.    -   93. A fusion protein comprising a rhizavidin protein and an S.        aureus peptide or polypeptide antigen, wherein the rhizavidin        protein comprises amino acids of SEQ ID NO: 1, or 85% sequence        identity to amino acids of SEQ ID NO: 1, and the S. aureus        peptide or polypeptide comprises a fragment of at least 20 amino        acids of Iron regulator surface protein B (IsdB), protein.    -   94. The fusion protein of paragraph 93, wherein the IsdB protein        comprises at least SEQ ID NO: 13 or a protein of at least 20        amino acids that has at least 85% sequence identity to SEQ ID        NO: 13.    -   95. A kit comprising:    -   (ii) a container comprising an immunogenic polysaccharide        cross-linked with a plurality of first affinity molecules; and    -   (iii) a container comprising a complementary affinity molecule        which associates with the first affinity molecule, wherein the        complementary affinity molecule associates with at least one S.        aureus antigen.    -   96. The kit of paragraph 95, further comprising a means to        attach the complementary affinity molecule to the antigen.    -   97. The kit of paragraph 95, further comprising at least one        co-stimulation factor.    -   98. The kit of paragraphs 95 to 97, further comprising a        cross-linking reagent which can be selected from the group        consisting of: CDAP (1-cyano-4-dimethylaminopyridinium        tetrafluoroborate), EDC (1-Ethyl-3-[3-dimethylaminopropyl]        carbodiimide hydrochloride), sodium cyanoborohydride, cyanogen        bromide, or ammonium bicarbonate/iodoacetic acid for linking the        co-factor to the polysaccharide.    -   99. The kit of paragraph 95, optionally comprising a container        comprising an expression vector for expressing an        antigen-affinity molecule fusion protein.    -   100. The kit of paragraph 99, wherein the expression vector can        optionally comprise a sequence for a linker peptide, wherein the        expression vector can express an antigen-affinity molecule        fusion protein comprising a linker peptide between the antigen        and the affinity molecule.    -   101. The kit of paragraph 95, wherein the antigen-affinity        molecule fusion protein is any of those selected from paragraphs        79-94.

EXAMPLES

The examples presented herein relate to methods to generate animmunogenic complex as described herein and methods and compositionsthereof. In particular, the examples relate to methods to produce a S.aureus multiple antigen presentation system (SA-MAPS) complex asdisclosed herein, and methods of use to generate an immune response in asubject.

Materials and Methods

Bacterial strains: S. aureus strains USA 300/TCH959 and ATCC 29213 wereoriginally purchased from ATCC. S. aureus strain USA 300 LAC wasprovided by BEI. Streptomycin-resistant USA 300 LAC strain (USA 300LAC^(strep)) was obtained by streaking USA 300 LAC strain on blood agarplate containing 0.5 g/L streptomycin and selecting a spontaneous mutantthe following morning.

Cloning and purification of S. aureus antigens. DNA sequences encodingClfA (221-559), ClfB (203-542), SdrD (246-682), IsdA (47-324), IsdB(48-477) or Hla (27-319) were amplified from S. aureus genomic DNA(purified from USA 300 TCH959 strain) via PCR and then cloned into apET-21b vector. A non-hemolytic toxoid of Hla was generated bysubstitution of residues Asp-Arg-Asp (aa209-211) (DRD) to Ala-Ala-Ala(AAA) using PCR. For rhizavidin fusion proteins, DNA sequences encodingSA antigens were inserted at the 3′ end of the gene encoding rhizavidinmoiety in a pET-21b vector. All constructs were transformed into E. coliBL21 (DE3) strain for expression. His-tagged recombinant proteins werepurified using Ni-nitrilotriacetic acid (NTA) affinity chromatography(Quagen) followed by size-exclusion chromatography using a Superdex 200column (GE lifescience). Purified proteins were stored at −80° C. untiluse.

Preparation of MAPS Complex. Type 1 pneumococcal capsular polysaccharide(referred to herein as CP1 or PS1 or SP PS1) was purchased from ATCC.Biotinylation of the polysaccharide was done as described previously.The SA-MAPS complex was assembled by incubation of biotinylatedimmunogenic polysaccharide with a mixture of rhizavidin fusions of S.aureus antigens at room temperature overnight. Typically, the rhizavidinfusion comprises 1:1 ratio of Rhizavidin: SA antigen. In someembodiments, the rhizavidin fusion comprises 1:2 ratio of Rhizavidin: SAantigen, in that the Rhizavidin protein can comprise 2 SA antigens,e.g., a Rhavi-A-A fusion protein, or a Rhavi-A-B fusion protein, where Ais one SA antigen, and B is a different SA antigen. In some embodiments,the rhizavidin fusion comprises 1:3 ratio of Rhizavidin: SA antigen, inthat the Rhizavidin protein can comprise 3 SA antigens, e.g., a Rhavi-A-A-A fusion protein, or a Rhavi-A-B-A fusion protein, or a Rhavi-A-B-Cfusion protein, where A is one SA antigen, and B is a different SAantigen, and C is a different SA antigen. The order of SA antigens A, Band C attached to the rhizavidin (Rhavi) protein of SEQ ID NO: 1 can bein any order, and in some embodiments, any one of A, B or C can be anon-SA antigen as disclosed herein.

In some embodiments, the input ratio of Rhizavidin fusion proteins topolysaccharide was 3:1 (w/w). The assembled complex was isolated bysize-exclusion chromatography. The fractions containing MAPS complexwere pooled and concentrated by ultrafiltration. The proteinconcentration in a MAPS complex was measured using a bicinchoninic acid(BCA) protein assay kit (Pierce). The incorporation of target antigenswas examined on a reduced SDS-PAGE gel.

Immunization and infection. Vaccines were prepared one day prior toimmunization. The antigens were diluted to the appropriate concentrationin saline, then mixed with aluminum hydroxide (Brenntag) and incubatedat 4° C. overnight with rotation. 4-6-week-old C57BL/6 wild type orμMT^(−/−)- female mice (Jackson Laboratories) received threesubcutaneous immunizations two weeks apart. Animals were bled two weekafter the last immunization for analysis of antibody and T-cellresponses. Mice were infected with S. aureus 7-10 days later.

Rabbit antisera against S. aureus antigens were generated at CocalicoBiologicals (Reamstown, Pa.). New Zealand White rabbits were given threeintramuscular immunizations, two weeks apart, with SA-MAPS vaccine. Serawere collected before the first immunization and two weeks after thelast immunization. For passive immunization, 8-week-old C57BL/6 femalemice received 200 μl of heat-inactivated pre- or post-vaccination rabbitsera one day prior to infection via intraperitoneal injection.

For all infections, S. aureus strains were streaked on blood agar platesand grown at 37° C. overnight. An overnight culture was then started byinoculating colonies into Tryptic soy broth (TSB, Sigma), shaking at 37°C. Cells were re-inoculated into fresh TSB medium at 1:100 dilution inthe morning and incubated at 37° C. with shaking for 3 hours. Bacterialcells were collected by centrifugation, washed twice with saline andadjusted to certain concentration in saline before infection.

Sepsis infection of S. aureus was performed using the ATCC 29213 strain.Mice were anesthetized with isoflurane and infected with 2-3×10⁷ CFU in100 μl via retro-orbital injection. Mice were monitored for sign ofillness for 14 days; any ill-appearing animal was immediately andhumanely euthanized.

In the dermonecrosis infection model, mice were anesthetized andinjected subcutaneously on the shaved lower back with 0.5-1×10⁷ CFU ofUSA300 TCH959 strain in 100 μl volume. Mice were monitored for 14 daysafter infection. Pictures of infected area were taken and the sizes ofdermonecrotic plaques/lesions were measured using ImageJ software.

In the skin abscess model, mice were shaved, anesthetized and infectedsubcutaneously with 2-5×10⁵ of USA300 TCH959 strain in 100 μl volume.Mice were then humanely euthanized on day 4 after infection. Abscesseswere dissected and homogenized by bead beater. Serial dilutions ofhomogenate were plated on mannitol salt plates and colonies were countedafter overnight culture at 37° C. Detection limit is 22.5 CFU. Inanimals that were abscess-free or for culture negative samples, theirCFU was set as the detection limit.

In the GI colonization model, mice were gently restrained and inoculatedintranasally with 5×10⁷ of USA 300 LAC^(strep) strain in 10 μl volume.Fecal pellets were collected on day 1 and day 7 after infection or asindicated. Samples were weighted, resuspended in sterile PBS at 0.1g/ml, homogenized and then passed through CellTrics 30 μm filter. Serialdilutions of the flow-through samples were plated on mannitol saltplates containing 0.5 g/L streptomycin and colonies were counted afterovernight culture at 37° C. Detection limit is 40 CFU. Forculture-negative samples, CFU was set as the detection limit.

Antibody and T-cell response analysis. Antigen-specific IgG antibody wasmeasured by ELISA using Immulon 2 HB 96-microwell plates (ThermoScientific) coated with individual recombinant S. aureus protein(non-rhizavidin fusion). The plates were washed with PBS containing0.05% Tween 20 (PBS-T) and then blocked with 1% BSA in PBS for 1 hour.After blocking, serial dilutions of mouse or rabbit sera were added andincubated for 2 hours, followed by 1 hour incubation with HRP-conjugatedsecondary antibody against mouse or rabbit IgG. The plates were thenwashed and developed with SureBlue TMB Microwell Peroxidase Substrate(KPL). 1 M HCl was used to terminate the reactions before the A_(450 nm)was analyzed using an ELISA reader. Antibody titers were expressed inarbitrary units relative to a standard serum.

For T-cell response analysis, 25 μl of heparinized blood were added to225 μl DMEM (BioWhittaker) containing 10% low-endotoxin defined FBS(Hyclone), 50 μM 2-mercaptoethanol (Sigma) and ciprofloxacin (10 μg/ml,Cellgro). The cultures were incubated at 37° C. for 6 days in thepresence of 2.5 μg/ml of the mixture of six S. aureus protein antigens(equal weight ratio, non-rhizavidin fusion). Supernatants were collectedfollowing centrifugation and analyzed by ELISA for INF-γ, IL-17A andIL-22 concentration (R&D Systems).

Hemolysis analysis. The hemolytic activity of wild type Hla, Hla209 andtheir rhizavidin fusions was measured as follows: 200 μl of heparinizedrabbit blood was washed with cold PBS three times. Red blood cells werethen resuspended in 10 ml of cold PBS (2% rabbit red blood cells) and100 μl of 2-fold serial dilution of Hla samples in PBS with 0.1% BSA,starting from 100 μg/ml, was added into a V-bottom 96-well plate beforethe addition of 100 μl of red blood cells to each well. PBS containing0.1% Triton X-100 was used as a positive control (100% hemolysis), andPBS with 0.1% BSA was used as a negative control (0% hemolysis). Theplate was incubated at 37° C. for 30 min and then subjected tocentrifugation at 800 g for 5 minutes. The supernatants were transferredinto a flat-bottom 96-well plate and the A₅4s. was measured by an ELISAreader. One hemolytic unit (HU) was defined as the activity that causes50% lysis of 1% rabbit red blood cells after 30 min incubation at 37° C.The activity of each Hla construct was expressed as the HU of 1 mg/ml ofpurified protein.

Statistical analysis. All statistical analyses were done using PRISM(version 5.01 for Windows, GraphPad Software, Inc). Antibody titer,cytokine release, size of lesions and CFU counts in abscesses or infeces were compared between groups using the Mann-Whitney U test.Differences in survival were analyzed by the Mantel-Cox test. Percentageof abscess formation was analyzed by the Fisher's exact test.

Example 1

It is well recognized that any single animal model of SA infection isunlikely to adequately represent the pathophysiology of disease inhumans; therefore, evaluation of any potential candidate in severalmodels would appear prudent. At the same time, the large number ofvirulence factors (including polysaccharides, surface proteins, andsecreted toxins produced by SA, may provide credence to the idea thatmultiple, genetically conserved antigens should be included in acandidate vaccine. Finally, a closer examination of mechanisms ofimmunity to SA in humans may also provide clues for an effective vaccinestrategy. Indeed, while humoral immunity plays a leading role in hostdefense against many bacterial or viral pathogens, it is unlikely thatantibodies are the only or even the primary factor for resistance to SA.Patients with B-cell deficiencies do not appear to be at significantlyincreased risk of SA infections, and individuals with high levels ofpre-existing SA-specific antibodies can still get infected by SA. On theother hand, a growing body of literature now suggests that T-cellimmunity, the other arm of acquired host defense, plays a critical rolein SA defense. Indeed, individuals with suppressed or impaired cellularimmunity, caused by high dose prednisone therapy, HIV infection,defective interferon-γ (IFN-γ) production, defective interleukin-17(IL-17) production, are at very high risk for SA infection andrecurrence. Moreover, in murine models, IFN-γ or IL-17A/F deficiency hasbeen shown to induce hyper-susceptibility to SA skin infections, andIL-17A deficiency in mice is also associated with prolonged nasalcarriage of SA. Therefore, an approach the combines both B- and T-cellacquired immunes responses to the organism may provide optimalprotection against this organism.

Based on these observations, the inventors designed and developed a SAantigen containing several conserved antigens, using a vaccine platformthat can elicit a broad range of immune responses, e.g., both B- andT-cell acquired responses. The inventors have previously developed asubunit vaccine platform, referred to as theMultiple-Antigen-Presenting-System (MAPS), which is disclosed in USApplication 2014/0154287, which is incorporated herein in its entiretyby reference. The MAPS generates affinity-coupled complexes of antigensthat can then induce broad B- and T-cell responses, and interestinglygenerated an immune response to both the immunogenic polypeptide as wellas the antigens.

Herein, the inventors prepared a multi-component SA subunit vaccineusing either a conventional approach (immunization with purifiedproteins alone) or proteins incorporated onto a scaffold using the MAPStechnology, as disclosed in US application 2014/0154287. The inventorsevaluated the immunogenicity of these two different vaccine approachesin mice, compared their protective efficacy in SA sepsis infection,dermonecrosis infection, skin abscess infection and gastrointestinal(GI) colonization models, and finally, studied the role ofantigen-specific antibodies and T-cell immunity against different typesof SA infection or colonization.

Preparation of vaccines. Six SA virulence factors that have beenpreviously studied and/or proposed as vaccine candidates were selected.Among them, α-hemolysin (Hla) is one of the most studied secreted toxinsof SA and has been shown to play an important role in early stages ofinvasive infection and skin infection. Clumping factor A (ClfA) andClumping factor B (ClfB), and serine-aspartate repeat protein D (SdrD)are cell-wall anchored adhesins that facilitate SA binding to theextracellular matrix or epithelial cells during colonization andinfection. ClfA has also been shown to be involved in acceleratedcleavage of complement 3b and thus result in decreasedcomplement-mediated phagocytosis of SA. Iron-regulated surface protein A(IsdA) and B (IsdB) function in iron-acquisition during SA infection.Antibodies against IsdA and IsdB have been shown to protect mice againstlethal intravenous challenge.

The selected antigens were cloned from the SA genome and thentransformed into E. coli for expression and purification. A detoxifiedHla mutant (Hla209) was generated by genetic substitution of residuesAsp-Arg-Asp (DRD) (209-211) to Ala-Ala-Ala (AAA) which resulted in morethan 700-fold reduction in hemolytic activity (FIG. 1A). A conventionalsubunit vaccine referred to herein as “SA-Mix”) was prepared by mixingall six recombinant proteins (e.g., ClfA (221-559); ClfB (203-542); SdrD(245-682); IsdA (47-324); IsdB (48-477) and Hla209(27-319)) at equalmolar ratio (see, FIG. 1B, upper panel). For SA-MAPS preparation, thetarget antigens were genetically fused to rhizavidin (rhavi), a dimericbiotin-binding protein identified in Rhizobium etli, as disclosed inU.S. Pat. No. 9,499,593 which is incorporated herein in its entirety byreference. The following Rhizavidin fusion proteins were generated,where the Rhizavidin protein of the fusion protein comprises SEQ ID NO:1; e.g., Rhavi-ClfA (221-559); Rhavi-ClfB (203-542); Rhavi-SdrD(245-682); Rhavi-IsdA (47-324); Rhavi-IsdB (48-477) andRhavi-Hla209(27-319). Rhavi-Hla209 fusion protein demonstrates furtherreduced hemolytic activity compared to Hla209 (FIG. 1A), due to thestoichiometric interference between the dimerization of rhavi and theheptameirzation of Hla which is required to initiate the hemolysis.SA-MAPS were assembled by affinity-coupling rhavi-SA antigens withbiotinylated type-1 pneumococcal capsular polysaccharide (FIG. 1B, lowerpanel), a polysaccharide not expected to contribute to protectionagainst SA infection. SDS-PAGE showed that all six target antigens wereincorporated into SA-MAPS complexes at approximately equal molar ratio(FIG. 1C).

Example 2

SA-MAPS is surprisingly significantly more immunogenic than the SA-Mixwith respect to both B- and T-cell responses to the target antigens.C57BL/6 mice received three subcutaneous administrations of adjuvantalone (Alum), SA-Mix or SA-MAPS vaccine. Serum IgG antibodies againsteach SA protein were measured two weeks after the last immunization.While both SA-Mix and SA-MAPS vaccine induced robust antibody responsesto the target antigens (FIG. 2A), the antibody titer in the SA-MAPSgroup was 2-6 fold higher than what was induced by SA-Mix cross all sixantigens (FIG. 2A), consistent with what the inventors have previouslyreported with a pneumococcal MAPS vaccine.

To examine antigen-specific T-cell responses, peripheral blood wascollected from the immunized animals and then stimulated in vitro with amixture of recombinant SA antigens (purified proteins without the rhavimoiety to ensure that the response is directed against the antigenrather than the affinity tag). Release of specific T-cell relatedcytokines in the culture supernatant was measured by ELISA afterstimulation. As shown in FIG. 2B, cells obtained from mice immunizedwith SA-Mix, did not produce any of the evaluated cytokines. Incontrast, the blood samples collected from SA-MAPS vaccinated animalsresponded to SA antigens and produced significant amount of IFN-γ,IL-17A and IL-22, implicating generation of Th1, Th17 and Th22 responsesby the MAPS vaccine.

Example 3

Vaccination with SA-MAPS provides broad protection against SA infectionand colonization. To better assess the potency of the SA-Mix and SA-MAPSvaccines, the inventors examined them in several SA infection orcolonization models. First, the inventors evaluated the performance ofthe SA-Mix and SA-MAPS vaccines in a mouse sepsis model: a highlyinvasive, systemic SA infection. Mice were injected retro-orbitally witha high dose of ATCC29213 strain which causes 60-90% death of naiveanimals within 7 days. In this model, vaccination with either SA-Mix orSA-MAPS significantly protected mice from SA-induced illness: whencompared to the control group which had 80% mortality after infection,the mortality of the SA-Mix or SA-MAPS group was reduced to 40% or 30%,respectively (FIG. 3A). While the survival rate was comparable betweenthe two vaccine groups, MAPS-vaccinated animals tended towards a delayedonset of illness compared to the SA-Mix group: 3 sick animals in theSA-MAPS group were found at 4.5, 10.5 days and 13 days after infectionrespectively, whereas all the sick animals in the control group or inthe SA-Mix group were identified within 1 week (2.5 to 6.5 days) afterinfection.

The inventors also evaluated two SSTI models: a dermonecrosis model inwhich the bacterial infection results in a large, measurable infectedplaque and the skin abscess model in which the infection is containedwithin an abscess and in which the bacterial burden can be quantified bydissection and harvest of the abscess and dilutional plating.Dermonecrosis was induced by subcutaneous injection of a USA300 strainwhich causes dermonecrotic plaques/lesions at the injection sites assoon as 2-3 days post infection. Lesions may further progress andmaximize in size in about 5-9 days post infection, often associated withpeeling skins and large open wounds (FIG. 3B, inset), and then begin toheal. In this model, both SA-Mix and SA-MAPS vaccine were highlyeffective in mitigating the infection. Compared to the control group inwhich 10 out of 10 animals developed dermonecrotic lesions afterinfection, only 2 mice in the SA-Mix group and 1 in the SA-MAPS grouphad any visible dermonecrosis (FIG. 3C). Besides the significantlyreduced incidence of lesions, the maximum lesion size on theMAPS-immunized mouse was also the smallest (<0.1 cm²) compared to whatwas found on the control animals (ranging from 0.17-1.2 cm²) or on theSA-Mix-vaccinated animals (0.4 and 0.89 cm²) (FIG. 3B).

In the skin abscess model, mice were infected subcutaneously with a muchlower inoculum of SA (approx. 40-50 fold lower than the dermonecrosisinfection) which leads to the formation of an enclosed subdermal abscessat the injection site, with no visible skin breakdown or damage (FIG.3D). Mice were sacrificed 4 days after infection and the abscesses wereisolated for CFU enumeration. Animals that received SA-MAPS vaccine werewell protected in the abscess model: 7 out of 10 mice in the SA-MAPSgroup were free of both abscess and bacteria on day 4 post-infection,whereas all mice in the control group developed abscesses (FIGS. 3D and3E). Interestingly, the SA-Mix group, which was as well protected as theSA-MAPS group in the dermonecrosis model, was neither protected againstabscess formation (FIG. 3D) nor did they display reduced bacterial loadin the abscesses (FIG. 3E), demonstrating that the mechanism ofprotection against these two types of SSTI was likely distinct and, inparticular, that T cell responses (which are lacking in the SA-Mixgroup) have a critical role in protection in the abscess model.

The last model the inventors examined was a GI colonization model, tomimic the non-pathogenic SA mucosal carriage condition. In this model,mice were inoculated intranasally with 5×10⁷ CFU of the USA300 LACstrain. One day later, fecal samples were collected from each animal andCFU of bacteria were enumerated and used as the baseline density of GIcarriage. For naive animals, 0.5-3×10⁵ CFU of SA could be recovered from1 gram of fecal samples (FIG. 4A) on day 1. The number of bacteria infeces decreases after the initial inoculation and then reaches arelatively stable colonization density between days 4 to 11post-inoculation, with densities ranging between 10³ and 10⁵ CFU pergram of feces (FIG. 4A). For vaccinated animals, fecal samples werecollected on day 1 (the baseline of GI carriage) and day 7post-inoculation for CFU analysis. Vaccination with SA-MAPSsignificantly reduced the density of SA GI colonization (FIG. 4B).Compared to baseline densities, fewer than 1% of bacteria (˜600 CFU pergram of feces) were recovered from the SA-MAPS vaccinated animals on day7 post-inoculation, whereas about 27% (˜18,000 CFU per gram of feces) ofthe original inoculum of SA still colonized the GI tracts of the controlanimals. Vaccination with SA-Mix, in contrast, did not have anyprotective impact on the clearance of SA carriage: about 84,000 CFU pergram of feces), or 29% of the baseline carriage, were found in theSA-Mix vaccinated animals on day 7 post-inoculation (FIGS. 4B and 4C).

Example 4

B- and T-cell immunity play different roles in protection againstdifferent types of SA infection or SA colonization. Thus, despitecomprising the same SA antigenic components, it was surprising thatthere was such marked differences in efficicies by the SA-Mix andSA-MAPS against SA infection or colonization. Importantly, vaccinationwith SA-MAPS provided broad protection in all four animal models tested,whereas SA-Mix provided protection only in the sepsis and dermonecrosismodels, but not skin abscess and GI colonization. As noted above, amajor and striking difference between these two vaccines is that SA-MAPSinduces not only antigen-specific antibodies, but also robust,antigen-specific T-cell responses. The inventors assessed if these Tcell responses explain the differences noted between the two vaccinationstrategies.

To test this hypothesis, the inventors began by evaluating thecontribution of antibodies using passive immunization. Anti-SA sera weregenerated by immunizing rabbits with SA-MAPS (FIG. 7). Pre-vaccinationrabbit sera were used as controls. Consistent with what we found withSA-Mix vaccination, which induces antibody-mediated protectionexclusively, passive immunization with rabbit anti-SA sera protectedmice in the sepsis and dermonecrosis infections. During sepsisinfection, mice that received anti-SA sera had a significantly improvedsurvival rate, at 50/6, compared to the control group which had 10%survival (FIG. 5A). Infusion with anti-SA sera was also able to mitigatethe findings in the dermonecrosis model. While most animals developedskin lesions, the size of lesions in the group that received immuneanti-sera was significantly smaller than in the control group (FIG. 5B).In contrast, we could not detect any impact of passive immunization inthe two other, less invasive models. In the abscess model, passiveimmunization with anti-SA sera showed no effect on either the formationof abscesses or the bacteria load in abscesses (FIG. 5C). Similarly,during GI colonization, with a comparable initial inoculation, theanimals that received anti-SA sera did not clear bacteria sooner thancontrols; in fact, they appeared to have an even higher density ofcolonization (about 4-fold higher in geometric mean) on day-7 comparedto the control group (FIG. 5D). Thus, while passive immunizationprovides significant protection against invasive disease anddermonecrosis, antibodies alone are ineffective in providing protectionin either the skin abscess or colonization models.

To evaluate to what degree protection in these models may be dependenton acquired T cell responses, the inventors evaluated SA-MAPS inμMT^(−/−)-mice, a mouse strain that is congenitally deficient inimmunoglobulins. Vaccination of μMT^(−/−)- mice with SA-MAPS induced noantigen-specific antibodies (FIG. 6A), but normal T-cell responses, asmeasured by the release of IFN-γ, IL-17 and IL-22 cytokines after invitro stimulation of the peripheral blood with SA antigens (FIG. 6B).Immunized μMT^(−/−)-mice were then challenged in our animal models. Ineither the sepsis or dermonecrosis infection model, against whichantigen-specific antibodies provide robust protection, the impact ofT-cell immunity was relatively minor. In the case sepsis infection,vaccination of μMT^(−/−) mice with SA-MAPS only slightly improved thesurvival rate (from 40% to 60%), with a trend towards slower developmentof illness, a phenomenon we have observed earlier in MAPS-vaccinated WTmice (FIG. 3A) but not in SA-Mix vaccinated WT mice or during passiveimmunization when only antibody responses were induced or present (FIGS.3A and 6C). In the case of dermonecrosis infection, antigen-specificT-cell immunity in the absence of antibodies was able to reduce the sizeof the lesions, especially in the first week of infection, compared tocontrol animals (FIG. 6D). In contrast, the contribution of T-cellimmunity was much more evident and in fact sufficient in either the skinabscess infection and GI colonization models, against which antibodieswere ineffective. Vaccination of μMT^(−/−)- mice with SA-MAPSsignificantly reduced abscess formation and bacterial burden (FIG. 6E).Furthermore, MAPS-induced T-cell immunity also facilitated the clearanceof SA in the GI tracts during colonization challenge. Seven dayspost-inoculation, mice in the MAPS-vaccinated group had greater than a10-fold reduction of geometric mean bacterial density in fecal samples,including complete clearance of SA in 4 out of 10 mice, whereas all theanimals in the control group remained colonized by SA and had only about2-fold reduction of geometric mean bacterial density in fecal samples(FIG. 6F).

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

1-101. (canceled)
 102. An immunogenic composition comprising: at least one immunogenic polysaccharide, at least two S. aureus peptide or polypeptide antigens, and at least one pair of affinity molecules, wherein the at least one pair of affinity molecules comprises a first affinity molecule and a second affinity molecule complementary to the first affinity molecule: wherein in each pair of affinity molecules: the first affinity molecule is associated with the at least one immunogenic polysaccharide, the second affinity molecule is associated with at least one of the S. aureus peptide or polypeptide antigens, and the first affinity molecule non-covalently associates with the second affinity molecule to link the S. aureus peptide or polypeptide antigen(s) and the immunogenic polysaccharide(s); and wherein the at least two S. aureus peptide or polypeptide antigens are each independently selected from the group consisting of: hemolysin (Hl), Clumping factor A (ClfA), Clumping factor B (ClfB), serine-aspartate repeat protein D (SdrD), serine-aspartate repeat protein E (SdrE), Iron regulator surface protein A (IsdA), Iron regulator surface protein B (IsdB), Leukotoxin D (LukD), and Leukotoxin E (LukE).
 103. The immunogenic composition of claim 102, wherein the at least two S. aureus peptide or polypeptide antigens are each independently selected from the group consisting of: HI, ClfA, ClfB, SdrD, IsdA, and IsdB.
 104. The immunogenic composition of claim 102, wherein the at least two S. aureus peptide or polypeptide antigens are each independently selected from the group consisting of: HI, ClfA, ClfB, and SdrD.
 105. The immunogenic composition of claim 102, comprising at least four S. aureus peptide or polypeptide antigens, wherein the at least four S. aureus peptide or polypeptide antigens are each independently selected from the group consisting of: hemolysin (Hl), Clumping factor A (ClfA), Clumping factor B (ClfB), serine-aspartate repeat protein D (SdrD), serine-aspartate repeat protein E (SdrE), Iron regulator surface protein A (IsdA), Iron regulator surface protein B (IsdB), Leukotoxin D (LukD), and Leukotoxin E (LukE).
 106. The immunogenic composition of claim 105, wherein the at least four S. aureus peptide or polypeptide antigens are or comprise HI, ClfA, ClfB, and SdrD.
 107. The immunogenic composition of claim 106, wherein the S. aureus Hl peptide or polypeptide antigen is or comprises a α-hemolysin (Hla), a β-hemolysin (Hlb) or a γ-hemolysin (Hl-gamma) from S. aureus.
 108. The immunogenic composition of claim 106, wherein the S. aureus Hl peptide or polypeptide antigen is or comprises wildtype Hla (WT Hla) or a Hla with a reduced hemolytic activity or a non-hemolytic Hla protein.
 109. The immunogenic composition of claim 106, wherein the S. aureus Hl peptide or polypeptide antigen is or comprises Hla (27-319).
 110. The immunogenic composition of claim 106, wherein the S. aureus ClfA peptide or polypeptide antigen is or comprises ClfA (221-559).
 111. The immunogenic composition of claim 106, wherein the S. aureus ClfB peptide or polypeptide antigen is or comprises ClfB (203-542).
 112. The immunogenic composition of claim 106, wherein the S. aureus SdrD peptide or polypeptide antigen is or comprises SdrD (246-682).
 113. The immunogenic composition of claim 108, wherein the S. aureus Hl peptide or polypeptide antigen is or comprises the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, or a polypeptide having an amino acid sequence with at least 85% sequence identity thereto.
 114. The immunogenic composition of claim 110, wherein the S. aureus ClfA peptide or polypeptide antigen is or comprises the amino acid sequence of SEQ ID NO: 3 or a polypeptide having an amino acid sequence with at least 85% sequence identity to the amino acid sequence of SEQ ID NO:
 3. 115. The immunogenic composition of claim 110, wherein the S. aureus ClfA peptide or polypeptide antigen is or comprises an antigenic fragment having at least 30 amino acids of the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least 30 amino acids in length that has at least 85% sequence identity to said antigenic fragment.
 116. The immunogenic composition of claim 111, wherein the S. aureus ClfB peptide or polypeptide antigen is or comprises the amino acid sequence of SEQ ID NO: 5 or a polypeptide having an amino acid sequence with at least 85% sequence identity to the amino acid sequence of SEQ ID NO:
 5. 117. The immunogenic composition of claim 111, wherein the S. aureus ClfB peptide or polypeptide antigen is or comprises an antigenic fragment having at least 30 amino acids of the amino acid sequence of SEQ ID NO: 4 or a polypeptide having at least 30 amino acids in length that has at least 85% sequence identity to said antigenic fragment.
 118. The immunogenic composition of claim 112, wherein the S. aureus SdrD peptide or polypeptide antigen is or comprises the amino acid sequence of SEQ ID NO: 7 or a polypeptide having an amino acid sequence with at least 85% sequence identity to the amino acid sequence of SEQ ID NO:
 7. 119. The immunogenic composition of claim 112, wherein the S. aureus SdrD peptide or polypeptide antigen is or comprises an antigenic fragment having at least 30 amino acids of the amino acid sequence of SEQ ID NO: 6 or a polypeptide having at least 30 amino acids in length that has at least 85% sequence identity to said antigenic fragment.
 120. The immunogenic composition of claim 102, wherein the first affinity molecule is or comprises biotin or a derivative or mimic molecule thereof.
 121. The immunogenic composition of claim 102, wherein the first affinity molecule is a biotin derivative, lipoic acid, HABA (hydroxyazobenzene-benzoic acid) and/or dimethyl-HABA or an amine-PEG3-biotin ((+)-biotinylation-3-6, 9-trixaundecanediamine).
 122. The immunogenic composition of claim 102, wherein the second affinity molecule is or comprises a biotin-binding protein.
 123. The immunogenic composition of claim 122, wherein the biotin-binding protein is or comprises rhizavidin, avidin, streptavidin, or a homologue or derivative thereof.
 124. The immunogenic composition of claim 123, wherein the rhizavidin is or comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that has at least 85% sequence identity to the amino acid sequence of SEQ ID NO:
 1. 125. The immunogenic composition of claim 102, wherein the first affinity molecule is cross-linked or covalently bonded to the immunogenic polysaccharide.
 126. The immunogenic composition of claim 102, wherein the second affinity molecule is fused to one or more of the at least two S. aureus peptide or polypeptide antigens to form a fusion protein.
 127. The immunogenic composition of claim 102, further comprising a flexible linker peptide attached to at least one of the at least two S. aureus peptide or polypeptide antigens, wherein the flexible linker peptide attaches the antigens to the second affinity molecule.
 128. The immunogenic composition of claim 102, wherein the immunogenic polysaccharide is a polysaccharide selected from the group consisting of: S. aureus, Vi polysaccharide, pneumococcal capsular polysaccharides, pneumococcal cell wall polysaccharide, Haemophilus influenzae Type b polysaccharide, Meningococcal polysaccharide, 0-antigens from Gram-negative bacteria, and other bacterial capsular or cell wall polysaccharides.
 129. The immunogenic composition of claim 102, wherein the immunogenic polysaccharide is selected from the group consisting of: type 1 capsular polysaccharide of Streptococcus pneumoniae, type 5 capsular polysaccharide of S. aureus, and type 8 capsular polysaccharide of S. aureus.
 130. The immunogenic composition of claim 102, further comprising at least one adjuvant.
 131. The immunogenic composition of claim 102 for use in any one or more of: a. as a diagnostic for exposure to a pathogen or immune threat, b. to prevent or treat an infection by S. aureus, or c. to prevent colonization of a subject by S. aureus.
 132. A method for inducing an immune response in a subject to S. aureus, comprising administering to the subject the immunogenic composition of claim
 102. 133. The method of claim 132, wherein the subject is a human subject.
 134. The method of claim 131, wherein the immune response is to the at least one immunogenic polysaccharide and at least one of the S. aureus peptide or polypeptide antigens,
 135. The method of claim 134, wherein the immune response is (i) an antibody or B-cell response, or (ii) an antibody or B-cell response and T-cell response.
 136. The method of claim 134, wherein the immune response is selected from any one of the following: a. an immune response that is a CD4+ T cell response, including Th1, Th2, or Th17 or Th22 response, or a CD8+ T cell response, or CD4+ and CD8+ T cell response, b. an antibody or B cell response to the at least one immunogenic polysaccharide and a CD4+ T cell response, including Th1, Th2, or Th17 or Th22 response, or a CD8+ T cell response, or CD4+/CD8+ T cell response to at least one of the S. aureus polypeptide antigens, c. an antibody or B cell response to the at least one immunogenic polysaccharide, and an antibody or B cell response and a CD4+ T cell response, including Th1, Th2, Th17 or Th22 responses, or a CD8+ T cell response, or CD4+/CD8+ T cell response to at least one of the S. aureus peptide or polypeptide antigens, or d. an antibody or B-cell response against at least one of the S. aureus peptide or polypeptide antigens which associates with the at least one immunogenic polysaccharide.
 137. The method of claim 132, wherein the immune response results in activation of INF-γ, IL-17A or IL-22 producing cells, or INF-γ, IL-17A and IL-22 producing cells. 